Methods for the detection, analysis and isolation of nascent proteins

ABSTRACT

This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)dyes.

This is a Continuation-In-Part of copending application(s) Ser. No.10/049,332 filed on Jun. 21, 2002 which is a U.S. entry of applicationPCT/US00/23233 filed on 23 Aug. 2000, which is a continuation in part ofU.S. Ser. No. 09/382,736, filed Aug. 25, 1999 now U.S. Pat. No.6,306,628 issued on Oct. 23, 2001.

FIELD OF THE INVENTION

This invention relates to non-radioactive markers that facilitate thedetection and analysis of nascent proteins translated within cellular orcell-free translation systems. Nascent proteins containing these markerscan be rapidly and efficiently detected, isolated and analyzed withoutthe handling and disposal problems associated with radioactive reagents.

BACKGROUND OF THE INVENTION

Cells contain organelles, macromolecules and a wide variety of smallmolecules. Except for water, the vast majority of the molecules andmacromolecules can be classified as lipids, carbohydrates, proteins ornucleic acids. Proteins are the most abundant cellular components andfacilitate many of the key cellular processes. They include enzymes,antibodies, hormones, transport molecules and components for thecytoskeleton of the cell.

Proteins are composed of amino acids arranged into linear polymers orpolypeptides. In living systems, proteins comprise over twenty commonamino acids. These twenty or so amino acids are generally termed thenative amino acids. At the center of every amino acid is the alphacarbon atom (Cα) which forms four bonds or attachments with othermolecules (FIG. 1). One bond is a covalent linkage to an amino group(NH₂) and another to a carboxyl group (COOH) which both participate inpolypeptide formation. A third bond is nearly always linked to ahydrogen atom and the fourth to a side chain which imparts variabilityto the amino acid structure. For example, alanine is formed when theside chain is a methyl group (—CH₃) and a valine is formed when the sidechain is an isopropyl group (—CH(CH₃)₂). It is also possible tochemically synthesize amino acids containing different side-chains,however, the cellular protein synthesis system, with rare exceptions,utilizes native amino acids. Other amino acids and structurally similarchemical compounds are termed non-native and are generally not found inmost organisms.

A central feature of all living systems is the ability to produceproteins from amino acids. Basically, protein is formed by the linkageof multiple amino acids via peptide bonds such as the pentapeptidedepicted in FIG. 1B. Key molecules involved in this process aremessenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules andribosomes (rRNA-protein complexes). Protein translation normally occursin living cells and in some cases can also be performed outside the cellin systems referred to as cell-free translation systems. In eithersystem, the basic process of protein synthesis is identical. Theextra-cellular or cell-free translation system comprises an extractprepared from the intracellular contents of cells. These preparationscontain those molecules which support protein translation and dependingon the method of preparation, post-translational events such asglycosylation and cleavages as well. Typical cells from which cell-freeextracts or in vitro extracts are made are Escherichia coli cells, wheatgerm cells, rabbit reticulocytes, insect cells and frog oocytes.

Both in vivo and in vitro syntheses involve the reading of a sequence ofbases on a mRNA molecule. The mRNA contains instructions for translationin the form of triplet codons. The genetic code specifies which aminoacid is encoded by each triplet codon. For each codon which specifies anamino acid, there normally exists a cognate tRNA molecule whichfunctions to transfer the correct amino acid onto the nascentpolypeptide chain. The amino acid tyrosine (Tyr) is coded by thesequence of bases UAU and UAC, while cysteine (Cys) is coded by UGU andUGC. Variability associated with the third base of the codon is commonand is called wobble.

Translation begins with the binding of the ribosome to mRNA (FIG. 2). Anumber of protein factors associate with the ribosome during differentphases of translation including initiation factors, elongation factorsand termination factors. Formation of the initiation complex is thefirst step of translation. Initiation factors contribute to theinitiation complex along with the mRNA and initiator tRNA (fmet and met)which recognizes the base sequence UAG. Elongation proceeds with chargedtRNAs binding to ribosomes, translocation and release of the amino acidcargo into the peptide chain. Elongation factors assist with the bindingof tRNAs and in elongation of the polypeptide chain with the help ofenzymes like peptidyl transferase. Termination factors recognize a stopsignal, such as the base sequence UGA, in the message terminatingpolypeptide synthesis and releasing the polypeptide chain and the mRNAfrom the ribosome.

The structure of tRNA is often shown as a cloverleaf representation(FIG. 3A). Structural elements of a typical tRNA include an acceptorstem, a D-loop, an anticodon loop, a variable loop and a TΨC loop.Aminoacylation or charging of tRNA results in linking the carboxylterminal of an amino acid to the 2′-(or 3′-) hydroxyl group of aterminal adenosine base via an ester linkage. This process can beaccomplished either using enzymatic or chemical methods. Normally aparticular tRNA is charged by only one specific native amino acid. Thisselective charging, termed here enzymatic aminoacylation, isaccomplished by aminoacyl tRNA synthetases. A tRNA which selectivelyincorporates a tyrosine residue into the nascent polypeptide chain byrecognizing the tyrosine UAC codon will be charged by tyrosine with atyrosine-aminoacyl tRNA synthetase, while a tRNA designed to read theUGU codon will be charged by a cysteine-aminoacyl tRNA synthetase. ThesetRNA synthetases have evolved to be extremely accurate in charging atRNA with the correct amino acid to maintain the fidelity of thetranslation process. Except in special cases where the non-native aminoacid is very similar structurally to the native amino acid, it isnecessary to use means other than enzymatic aminoacylation to charge atRNA.

Molecular biologists routinely study the expression of proteins that arecoded for by genes. A key step in research is to express the products ofthese genes either in intact cells or in cell-free extracts.Conventionally, molecular biologists use radioactively labeled aminoacid residues such as ³⁵S-methionine as a means of detecting newlysynthesized proteins or so-called nascent proteins. These nascentproteins can normally be distinguished from the many other proteinspresent in a cell or a cell-free extract by first separating theproteins by the standard technique of gel electrophoresis anddetermining if the proteins contained in the gel possess the specificradioactively labeled amino acids. This method is simple and relies ongel electrophoresis, a widely available and practiced method. It doesnot require prior knowledge of the expressed protein and in general doesnot require the protein to have any special properties. In addition, theprotein can exist in a denatured or unfolded form for detection by gelelectrophoresis. Furthermore, more specialized techniques such asblotting to membranes and coupled enzymatic assays are not needed.Radioactive assays also have the advantage that the structure of thenascent protein is not altered or can be restored, and thus, proteinscan be isolated in a functional form for subsequent biochemical andbiophysical studies.

Radioactive methods suffer from many drawbacks related to theutilization of radioactively labeled amino acids. Handling radioactivecompounds in the laboratory always involves a health risk and requiresspecial laboratory safety procedures, facilities and detailed recordkeeping as well as special training of laboratory personnel. Disposal ofradioactive waste is also of increasing concern both because of thepotential risk to the public and the lack of radioactive waste disposalsites. In addition, the use of radioactive labeling is time consuming,in some cases requiring as much as several days for detection of theradioactive label. The long time needed for such experiments is a keyconsideration and can seriously impede research productivity. Whilefaster methods of radioactive detection are available, they areexpensive and often require complex image enhancement devices.

The use of radioactive labeled amino acids also does not allow for asimple and rapid means to monitor the production of nascent proteinsinside a cell-free extract without prior separation of nascent frompreexisting proteins. However, a separation step does not allow for theoptimization of cell-free activity. Variables including theconcentration of ions and metabolites and the temperature and the timeof protein synthesis cannot be adjusted.

Radioactive labeling methods also do not provide a means of isolatingnascent proteins in a form which can be further utilized. The presenceof radioactivity compromises this utility for further biochemical orbiophysical procedures in the laboratory and in animals. This is clearin the case of in vitro expression when proteins cannot be readilyproduced in vivo because the protein has properties which are toxic tothe cell. A simple and convenient method for the detection and isolationof nascent proteins in a functional form could be important in thebiomedical field if such proteins possessed diagnostic or therapeuticproperties. Recent research has met with some success, but these methodshave had numerous drawbacks.

Radioactive labeling methods also do not provide a simple and rapidmeans of detecting changes in the sequence of a nascent protein whichcan indicate the presence of potential disease causing mutations in theDNA which code for these proteins or fragments of these proteins.Current methods of analysis at the protein level rely on the use of gelelectrophoresis and radioactive detection which are slow and notamenable to high throughput analysis and automation. Such mutations canalso be detected by performing DNA sequence analysis on the gene codingfor a particular protein or protein fragment. However, this requireslarge regions of DNA to be sequenced, which is time-consuming andexpensive. The development of a general method which allows mutations tobe detected at the nascent protein level is potentially very importantfor the biomedical field.

Radioactive labeling methods also do not provide a simple and rapidmeans of studying the interaction of nascent proteins with othermolecules including compounds which might be have importance aspotential drugs. If such an approach were available, it could beextremely useful for screening large numbers of compounds against thenascent proteins coded for by specific genes, even in cases where thegenes or protein has not yet been characterized. In current technology,which is based on affinity electrophoresis for screening of potentialdrug candidates, both in natural samples and synthetic libraries,proteins must first be labeled uniformly with a specific marker whichoften requires specialized techniques including isolation of the proteinand the design of special ligand markers or protein engineering.

Special tRNAs, such as tRNAs which have suppressor properties,suppressor tRNAs, have been used in the process of site-directednon-native amino acid replacement (SNAAR) (C. Noren et al., Science244:182-188, 1989). In SNAAR, a unique codon is required on the mRNA andthe suppressor tRNA, acting to target a non-native amino acid to aunique site during the protein synthesis (PCT WO90/05785). However, thesuppressor tRNA must not be recognizable by the aminoacyl tRNAsynthetases present in the protein translation system (Bain et al.,Biochemistry 30:5411-21, 1991). Furthermore, site-specific incorporationof non-native amino acids is not suitable in general for detection ofnascent proteins in a cellular or cell-free protein synthesis system dueto the necessity of incorporating non-sense codons into the codingregions of the template DNA or the mRNA.

Products of protein synthesis may also be detected by using antibodybased assays. This method is of limited use because it requires that theprotein be folded into a native form and also for antibodies to havebeen previously produced against the nascent protein or a known proteinwhich is fused to the unknown nascent protein. Such procedures are timeconsuming and again require identification and characterization of theprotein. In addition, the production of antibodies and amino acidsequencing both require a high level of protein purity.

In certain cases, a non-native amino acid can be formed after the tRNAmolecule is aminoacylated using chemical reactions which specificallymodify the native amino acid and do not significantly alter thefunctional activity of the aminoacylated tRNA (Promega TechnicalBulletin No. 182; tRNA^(nscend)™: Non-radioactive Translation DetectionSystem, September 1993). These reactions are referred to aspost-aminoacylation modifications. For example, the ε-amino group of thelysine linked to its cognate tRNA (tRNA^(LYS)), could be modified withan amine specific photoaffinity label (U. C. Krieg et al., Proc. Natl.Acad. Sci. USA 83:8604-08, 1986). These types of post-aminoacylationmodifications, although useful, do not provide a general means ofincorporating non-native amino acids into the nascent proteins. Thedisadvantage is that only those non-native amino acids that arederivatives of normal amino acids can be incorporated and only a fewamino acid residues have side chains amenable to chemical modification.More often, post-aminoacylation modifications can result in the tRNAbeing altered and produce a non-specific modification of the α-aminogroup of the amino acid (e.g. in addition to the ε-amino group) linkedto the tRNA. This factor can lower the efficiency of incorporation ofthe non-native amino acid linked to the tRNA. Non-specific,post-aminoacylation modifications of tRNA structure could alsocompromise its participation in protein synthesis. Incomplete chainformation could also occur when the α-amino group of the amino acid ismodified.

In certain other cases, a nascent protein can be detected because of itsspecial and unique properties such as specific enzymatic activity,absorption or fluorescence. This approach is of limited use since mostproteins do not have special properties with which they can be easilydetected. In many cases, however, the expressed protein may not havebeen previously characterized or even identified, and thus, itscharacteristic properties are unknown.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides methods forthe labeling, detection, quantitation, analysis and isolation of nascentproteins produced in a cell-free or cellular translation system withoutthe use of radioactive amino acids or other radioactive labels. Oneembodiment of the invention is directed to methods for detecting nascentproteins translated in a translation system. A tRNA molecule isaminoacylated with a fluorescent marker to create a misaminoacylatedtRNA. The misaminoacylated, or charged, tRNA can be formed by chemical,enzymatic or partly chemical and partly enzymatic techniques which placea fluorescent marker into a position on the tRNA molecule from which itcan be transferred into a growing peptide chain. Markers may comprisenative or non-native amino acids with fluorescent moieties, amino acidanalogs or derivatives with fluorescent moieties, detectable labels,coupling agents or combinations of these components with fluorescentmoieties. The misaminoacylated tRNA is introduced to the translationsystem such as a cell-free extract, the system is incubated and thefluorescent marker incorporated into nascent proteins.

It is not intended that the present invention be limited to the natureof the particular fluorescent moiety. A variety of fluorescent compoundsare contemplated, including fluorescent compounds that have beenderivatized (e.g. with NHS) to be soluble (e.g. NHS-derivatives ofcoumarin). Nonetheless, compared to many other fluorophores with highquantum yields, several BODIPY compounds and reagents have beenempirically found to have the additional important and unusual propertythat they can be incorporated with high efficiency into nascent proteinsfor both UV and visible excited fluorescence detection. These methodsutilizing fluorescent moieties may be used to detect, isolate andquantitate such nascent proteins as recombinant gene products, genefusion products, truncated proteins caused by mutations in human genes,enzymes, cytokines, hormones, immunogenic proteins, human proteins,carbohydrate and lipid binding proteins, nucleic acid binding proteins,viral proteins, bacterial proteins, parasitic proteins and fragments andcombinations thereof.

Another embodiment of the invention is directed to methods for labelingnascent proteins at their amino terminus. An initiator tRNA molecule,such as methionine initiator tRNA or formylmethionine-initiator tRNA ismisaminoacylated with a fluorescent moiety (e.g. a BODIPY moiety) andintroduced to a translation system. The system is incubated and markeris incorporate at the amino terminus of the nascent proteins. Nascentproteins containing marker can be detected, isolated and quantitated.Markers or parts of markers may be cleaved from the nascent proteinswhich substantially retain their native configuration and arefunctionally active.

Thus, the present invention contemplates compositions, methods andsystems. In terms of compositions, the present invention specificallycontemplates a tRNA molecule misaminoacylated with a BODIPY marker.

In one embodiment, the present invention contemplates a method,comprising: a) providing a tRNA molecule and a BODIPY marker; and b)aminoacylating said tRNA molecule with said BODIPY marker to create amisaminoacylated tRNA. In a particular embodiment, the method furthercomprises c) introducing said misaminoacylated tRNA into a translationsystem under conditions such that said marker is incorporated into anascent protein. In yet another embodiment, the method further comprisesd) detecting said nascent protein containing said marker. In stillanother embodiment, the method further comprises e) isolating saiddetected nascent protein.

The present invention contemplates aminoacylation of the tRNA moleculeby chemical or enzymatic misaminoacylation. The present invention alsocontemplates embodiments wherein two or more different misaminoacylatedtRNAs are introduced into the translation system. In a preferredembodiment, the nascent protein detected (by virtue of the incorporatedmarker) is functionally active.

It is not intended that the present invention be limited by theparticular nature of the nascent protein. In one embodiment, the presentinvention contemplates a method for detecting nascent proteins which areconjugated to the mRNA message which codes for all or part of thenascent protein. In general, a variety of modifications of the nascentprotein are envisioned including post-translational modifications,proteolysis, attachment of an oligonucleotide through a puromycin linkerto the C-terminus of the protein, and interaction of the nascent proteinwith other components of the translation system including those whichare added exogenously.

It is not intended that the present invention be limited by theparticular nature of the tRNA molecule. In one embodiment, the tRNAmolecule is an initiator tRNA molecule. In another embodiment, the tRNAmolecule is a suppressor tRNA molecule.

The present invention also contemplates kits. In one embodiment, the kitcomprises a) a first containing means (e.g. tubes, vials, etc)containing at least one component of a protein synthesis system; and b)a second containing means containing a misaminoacylated tRNA, whereinsaid tRNA is misaminoacylated with a BODIPY marker. Such kits mayinclude initiator tRNA and/or suppressor tRNA. Importantly, the kit isnot limited to the particular components of said protein synthesissystem; a variety of components are contemplated (e.g. ribosomes).

Another embodiment of the invention is directed to methods for detectingnascent proteins translated in a translation system. A tRNA molecule isaminoacylated with one component of a binary marker system. Themisaminoacylated, or charged, tRNA can be formed by chemical, enzymaticor partly chemical and partly enzymatic techniques which place acomponent of a binary marker system into a position on the tRNA moleculefrom which it can be transferred into a growing peptide chain. Thecomponent of the binary marker system may comprise native or non-nativeamino acids, amino acid analogs or derivatives, detectable labels,coupling agents or combinations of these components. Themisaminoacylated tRNA is introduced to the translation system such as acell-free extract, the system is incubated and the marker incorporatedinto nascent proteins. The second component of the binary marker systemis then introduced making the first component incorporated into thenascent protein specifically detectable. These methods may be used todetect, isolate and quantitate such nascent proteins as recombinant geneproducts, gene fusion products, enzymes, cytokines, hormones,immunogenic proteins, human proteins, carbohydrate and lipid bindingproteins, nucleic acid binding proteins, viral proteins, bacterialproteins, parasitic proteins and fragments and combinations thereof.

It is not intended that the present invention be limited to a particulartranslation system. In one embodiment, a cell-free translation system isselected from the group consisting of Escherichia coli lysates, wheatgerm extracts, insect cell lysates, rabbit reticulocyte lysates, frogoocyte lysates, dog pancreatic lysates, human cell lysates, mixtures ofpurified or semi-purified translation factors and combinations thereof.It is also not intended that the present invention be limited to theparticular reaction conditions employed. However, typically thecell-free translation system is incubated at a temperature of betweenabout 25° C. to about 45° C. The present invention contemplates bothcontinuous flow systems or dialysis systems.

Another embodiment of the invention is directed to methods for thedetection of nascent proteins translated in a cellular or cell-freetranslation system using non-radioactive markers which have detectableelectromagnetic spectral properties. As before, a non-radioactive markeris misaminoacylated to a tRNA molecule and the misaminoacylated tRNA isadded to the translation system. The system is incubated to incorporatemarker into the nascent proteins. Nascent proteins containing marker canbe detected from the specific electromagnetic spectral property of themarker. Nascent proteins can also be isolated by taking advantage ofunique properties of these markers or by conventional means such aselectrophoresis, gel filtration, high-pressure or fast-pressure liquidchromatography, affinity chromatography, ion exchange chromatography,chemical extraction, magnetic bead separation, precipitation orcombinations of these techniques.

Another embodiment of the invention is directed to the synthesis ofnascent proteins containing markers which have reporter properties whenthe reporter is brought into contact with a second agent. Reportermarkers are chemical moieties which have detectable electromagneticspectral properties when incorporated into peptides and whose spectralproperties can be distinguished from unincorporated markers and markersattached to tRNA molecules. As before, tRNA molecules aremisaminoacylated, this time using reported markers. The misaminoacylatedtRNAs are added to a translation system and incubated to incorporatemarker into the peptide. Reporter markers can be used to follow theprocess of protein translation and to detect and quantitate nascentproteins without prior isolation from other components of the proteinsynthesizing system.

Another embodiment of the invention is directed to compositionscomprised of nascent proteins translated in the presence of markers,isolated and, if necessary, purified in a cellular or cell-freetranslation system. Compositions may further comprise a pharmaceuticallyacceptable carrier and be utilized as an immunologically activecomposition such as a vaccine, or as a pharmaceutically activecomposition such as a drug, for use in humans and other mammals.

Another embodiment of the invention is directed to methods for detectingnascent proteins translated in a translation system by using massspectrometry. A non-radioactive marker of known mass is misaminoacylatedto a tRNA molecule and the misaminoacylated tRNA is added to thetranslation system. The system is incubated to incorporate the massmarker into the nascent proteins. The mass spectrum of the translationsystem is then measured. The presence of the nascent protein can bedirectly detected by identifying peaks in the mass spectrum of theprotein synthesis system which correspond to the mass of the unmodifiedprotein and a second band at a higher mass which corresponds to the massof the nascent protein plus the modified amino acid containing the massof the marker. When the mass marker is photocleavable, the assignment ofthe second band to a nascent protein containing the mass marker can beverified by removing the marker with light.

Another embodiment of the invention is directed to methods for detectingnascent proteins with mutations which are translated in a translationsystem. RNA or DNA coding for the protein which may contain a possiblemutation is added to the translation system. The system is incubated tosynthesize the nascent proteins. The nascent protein is then separatedfrom the translation system using an affinity marker. In one embodimentthe affinity marker is located at or close to the N-terminal end of theprotein while in another embodiment the affinity marker can bedistributed randomly throughout the sequence of the protein. The proteinis then analyzed for the presence of a detectable marker located at orclose to the N-terminal of the protein (N-terminal marker). A separatemeasurement is then made on a sequence dependent detectable markerlocated at or close to the C-terminal end of the protein (C-terminalmarker). A comparison is then made of the level of incorporation of theN-terminal and C-terminal markers in the nascent protein. It is notintended that the present invention be limited by the nature of the N-and C-terminal markers, or the type of affinity marker utilized. Avariety of markers are contemplated. In one embodiment, the affinitymarker comprises an epitope recognized by an antibody or other bindingmolecule. In another embodiment, the affinity marker is biotin and isdistributed randomly on lysine residues. In one embodiment, theN-terminal marker comprises a fluorescent marker (e.g. a BODIPY marker),while the C-terminal marker comprises a metal binding region (e.g. Histag).

The present invention contemplates a variety of methods wherein thethree markers (e.g. the N- and C-terminal markers and the affinitymarkers) are introduced into a nascent protein. In one embodiment, themethod comprises: a) providing i) a misaminoacylated initiator tRNAmolecule which only recognizes the first AUG codon that serves toinitiate protein synthesis, said misaminoacylated initiator tRNAmolecule comprising a first marker, and ii) a nucleic acid templateencoding a protein, said protein comprising a C-terminal marker and (insome embodiments) an affinity marker; b) introducing saidmisaminoacylated initiator tRNA to a translation system comprising saidtemplate under conditions such that a nascent protein is generated, saidprotein comprising said first marker, said C-terminal marker and (insome embodiments) said affinity marker. In one embodiment, the methodfurther comprises, after step b), isolating said nascent protein.

In another embodiment, the method comprises: a) providing i) amisaminoacylated initiator tRNA molecule which only recognizes the firstAUG codon that serves to initiate protein synthesis, saidmisaminoacylated initiator tRNA molecule comprising a first marker, andii) a nucleic acid template encoding a protein, said protein comprisinga C-terminal marker and (in some embodiments) an affinity marker; b)introducing said misaminoacylated initiator tRNA to a translation systemcomprising said template under conditions such that a nascent protein isgenerated, said protein comprising said first marker at the N-terminusof said protein, a C-terminal marker, and (in some embodiments) saidaffinity marker adjacent to said first marker. In one embodiment, themethod further comprises, after step b), isolating said nascent protein.

In yet another embodiment, the method comprises: a) providing i) amisaminoacylated tRNA molecule which only recognizes the first codondesigned to serve to initiate protein synthesis, said misaminoacylatedinitiator tRNA molecule comprising a first marker, and ii) a nucleicacid template encoding a protein, said protein comprising a C-terminalmarker and (in some embodiments) an affinity marker; b) introducing saidmisaminoacylated initiator tRNA to a translation system comprising saidtemplate under conditions such that a nascent protein is generated, saidprotein comprising said first marker, said C-terminal marker and (insome embodiments) said affinity marker. In one embodiment, the methodfurther comprises, after step b), isolating said nascent protein.

In still another embodiment, the method comprises: a) providing i) amisaminoacylated tRNA molecule which only recognizes the first codondesigned to serve to initiate protein synthesis, said misaminoacylatedinitiator tRNA molecule comprising a first marker, and ii) a nucleicacid template encoding a protein, said protein comprising a C-terminalmarker and (in some embodiments) an affinity marker; b) introducing saidmisaminoacylated initiator tRNA to a translation system comprising saidtemplate under conditions such that a nascent protein is generated, saidprotein comprising said first marker at the N-terminus of said protein,a C-terminal marker, and (in some embodiments) said affinity markeradjacent to said first marker. In one embodiment, the method furthercomprises, after step b), isolating said nascent protein.

The present invention also contemplates embodiments where only twomarkers are employed (e.g. a marker at the N-terminus and a marker atthe C-terminus). In one embodiment, the nascent protein isnon-specifically bound to a solid support (e.g. beads, microwells,strips, etc.), rather than by the specific interaction of an affinitymarker. In this context, “non-specific” binding is meant to indicatethat binding is not driven by the uniqueness of the sequence of thenascent protein. Instead, binding can be by charge interactions as wellas hydrophilic or hydrophobic interactions. In one embodiment, thepresent invention contemplates that the solid support is modified (e.g.functionalized to change the charge of the surface) in order to capturethe nascent protein on the surface of the solid support. In oneembodiment, the solid support is poly-L-lysine coated. In yet anotherembodiment, the solid support is nitrocellulose (e.g. strips,nitrocellulose containing microwells, etc.) or alternativelypolystyrene. Regardless of the particular nature of the solid support,the present invention contemplates that the nascent protein containingthe two markers is captured under conditions that permit the readydetection of the markers.

In both the two marker and three marker embodiments described above, thepresent invention contemplates that one or more of the markers will beintroduced into the nucleic acid template by primer extension or PCR. Inone embodiment, the present invention contemplates a primer comprising(on or near the 5′-end) a promoter, a ribosome binding site (“RBS”), anda start codon (e.g. ATG), along with a region of complementarity to thetemplate. In another embodiment, the present invention contemplates aprimer comprising (on or near the 5′-end) a promoter, a ribosome bindingsite (“RBS”), a start codon (e.g. ATG), a region encoding an affinitymarker, and a region of complementarity to the template. It is notintended that the present invention be limited by the length of theregion of complementarity; preferably, the region is greater than 8bases in length, more preferably greater than 15 bases in length, andstill more preferably greater than 20 bases in length.

It is also not intended that the present invention be limited by theribosome binding site. In one embodiment, the present inventioncontemplates primers comprising the Kozak sequence, a string ofnon-random nucleotides (consensus sequence 5′-GCCA/GCCATGG-3′) (SEQ IDNO:1) which are present before the translation initiating first ATG inmajority of the mRNAs which are transcribed and translated in eukaryoticcells. See M. Kozak, Cell 44:283-292 (1986). In another embodiment, thepresent invention contemplates a primer comprising the the prokaryoticmRNA ribosome binding site, which usually contains part or all of apolypurine domain UAAGGAGGU (SEQ ID NO:2) known as the Shine-Dalgarno(SD) sequence found just 5′ to the translation initiation codon: mRNA5′-UAAGGAGGU-N₅₋₁₀-AUG. (SEQ ID NO:3)

For PCR, two primers are used. In one embodiment, the present inventioncontemplates as the forward primer a primer comprising (on or near the5′-end) a promoter, a ribosome binding site (“RBS”), and a start codon(e.g. ATG), along with a region of complementarity to the template. Inanother embodiment, the present invention contemplates as the forwardprimer a primer comprising (on or near the 5′-end) a promoter, aribosome binding site (“RBS”), a start codon (e.g. ATG), a regionencoding an affinity marker, and a region of complementarity to thetemplate. The present invention contemplates that the reverse primer, inone embodiment, comprises (at or near the 5′-end) one or more stopcodons and a region encoding a C-terminus marker (such as a HIS-tag).

The present invention also contemplates embodiments where the affinitymarker is introduced through a misaminoacylated tRNA. In one embodimentthe misaminoacylated tRNA only recognizes a codon which codes for aparticular amino acid such as a codon for lysine. In this case, theaffinity marker is incorporated randomly throughout the proteinsequence. In another embodiment, more than one misaminoacylated tRNA isutilized. In this case, the affinity marker may be randomly distributedthroughout the protein sequence at more than a single amino acid such aslysine or tyrosine. In another embodiment the misaminoacylated tRNA is asuppressor tRNA and incorporates the affinity marker at a specificposition in the protein sequence.

Another embodiment of the invention is directed to methods for detectingby electrophoresis (e.g. capillary electrophoresis) the interaction ofmolecules with nascent proteins which are translated in a translationsystem. A tRNA misaminoacylated with a detectable marker is added to theprotein synthesis system. The system is incubated to incorporate thedetectable marker into the nascent proteins. One or more specificmolecules are then combined with the nascent proteins (either before orafter isolation) to form a mixture containing nascent proteins/moleculeconjugates. Aliquots of the mixture are then subjected to capillaryelectrophoresis. Nascent proteins/molecule conjugates are identified bydetecting changes in the electrophoretic mobility of nascent proteinswith incorporated markers.

The present invention also contemplates using the above describedmethods and compositions in a method for drug/proteome screening. Forexample, the present invention contemplates a method of identifying aprotein or portion of a protein which interacts with a ligand (such as adrug), which method comprises the steps of preparing a cDNA library froma cell that expresses said desired protein; inserting said cDNA libraryinto an expression vector, thereby forming an expression cDNA library;transforming said expression library into bacterial cells and culturingsaid bacterial cells to produce individual bacterial colonies, whereineach colony contains a member of said expression library; collectingpools of a predetermined number of said individual bacterial colonies;isolating cDNA from said pools of bacterial colonies; expressingproteins (or portions of proteins) encoded by said cDNAs; andidentifying a desired protein (or portion thereof) and the cDNA encodingsaid desired protein (or portion); wherein pools of approximately 50individual bacterial colonies are collected (more preferably less than50, still more preferably less than 40, still more preferably less than30, and most preferably 25 or less, but more than 10), wherein eachcolony contains a member of an expression cDNA library, thereby formingcDNA pools; and wherein proteins (or portions thereof) encoded by thecDNA in said cDNA pools are expressed in an in vitrotranscription/translation system in a common reaction mixture, andwherein a desired protein (or portion thereof) is identified from saidreaction mixture.

Another aspect of the present invention contemplates an oligonucleotide,comprising a 5′ portion, a middle portion contiguous with said 5′portion, and a 3′ portion contiguous with said middle portion, whereini) said 5′ portion comprises a sequence corresponding to a promoter, ii)said middle portion comprises a sequence corresponding to a ribosomebinding site, a start codon, and a sequence coding for an epitopemarker, wherein said epitope marker consists of a portion of the p53amino acid sequence or variant thereof, and iii) said 3′ portioncomprises a sequence complementary to a portion of the APC gene (oranother gene whose truncated products are associated with disease, i.e.a “disease related gene”). In one embodiment, said oligonucleotide isless than one hundred bases in length. In another embodiment, saidoligonucleotide has the sequence set forth in SEQ ID NO: 22. In oneembodiment, said 5′ portion is between ten and forty bases in length(preferably between eight and sixty bases in length, and more preferablybetween fifteen and thirty bases in length). In one embodiment, saidmiddle portion is between ten and three thousand bases in length(preferably between eight and sixty bases in length, and more preferablybetween fifteen and thirty bases in length). In one embodiment, said 3′portion is between ten and three thousand bases in length (preferablybetween eight and sixty bases in length, and more preferably betweenfifteen and thirty bases in length). In one embodiment, said sequencecomplementary to the portion of the APC gene is greater than 15 bases inlength. In another embodiment, said sequence complementary to theportion of the APC gene is greater than 20 bases in length. In oneembodiment, said sequence coding for an epitope marker codes for theamino acid sequence selected from SEQ ID NOS:24-38. In anotherembodiment, said sequence coding for an epitope marker codes for theamino acid sequence selected from SEQ ID NOS: 39-46.

Another aspect of the present invention contemplates an oligonucleotide,comprising a 5′ portion, a middle portion contiguous with said 5′portion, and a 3′ portion contiguous with said middle portion, whereini) said 5′ portion comprises at least one stop codon, ii) said middleportion comprises a sequence encoding for an epitope marker, whereinsaid epitope marker consists of a portion of the VSV-G amino acidsequence or variant thereof, and iii) said 3′ portion comprises asequence complementary to a portion of the APC gene (or another genewhose truncated products are associated with disease). In oneembodiment, said oligonucleotide is less than one hundred bases inlength. In another embodiment, said oligonucleotide has the sequence setforth in SEQ ID NO: 23. In one embodiment, said 5′ portion is betweenten and forty bases in length (preferably between eight and sixty basesin length, and more preferably between fifteen and thirty bases inlength). In one embodiment, said middle portion is between ten and fortybases in length (preferably between eight and sixty bases in length, andmore preferably between fifteen and thirty bases in length). In oneembodiment, said 3′ portion is between ten and forty bases in length(preferably between eight and sixty bases in length, and more preferablybetween fifteen and thirty bases in length). In one embodiment, saidsequence complementary to the portion of the APC gene is greater than 15bases in length. In another embodiment, said sequence complementary tothe portion of the APC gene is greater than 20 bases in length. In oneembodiment, said sequence coding for an epitope marker codes for theamino acid sequence selected from SEQ ID NOS:24-38. In anotherembodiment, said sequence coding for an epitope marker codes for theamino acid sequence selected from SEQ ID NOS: 39-46.

Another aspect of the present invention contemplates a kit, comprising:a) a first oligonucleotide comprising a 5′ portion, a middle portioncontiguous with said 5′ portion, and a 3′ portion contiguous with saidmiddle portion, wherein i) said 5′ portion comprises a sequencecorresponding to a promoter, ii) said middle portion comprises asequence corresponding to a ribosome binding site, a start codon, and asequence coding for a first epitope marker, and iii) said 3′ portioncomprises a sequence complementary to a first portion of the APC gene(or other disease related gene); b) a second oligonucleotide comprisinga 5′ portion, a middle portion contiguous with said 5′ portion, and a 3′portion contiguous with said middle portion, wherein i) said 5′ portioncomprises at least one stop codon, ii) said middle portion comprises asequence encoding for a second epitope marker, and iii) said 3′ portioncomprises a sequence complementary to a second portion of the APC gene(or other disease related gene), wherein either said first epitopemarker or said second epitope marker consist of a portion of the p53amino acid sequence or variant thereof. In one embodiment, said sequencecoding for said first epitope marker codes for the amino acid sequenceselected from SEQ ID NOS: 39-46. In one embodiment, said sequence codingfor said second epitope marker codes for the amino acid sequenceselected from SEQ ID NOS: 24-38. In one embodiment, said firstoligonucleotide has the sequence set forth in SEQ ID NO: 22. In oneembodiment, said second oligonucleotide has the sequence set forth inSEQ ID NO: 23. In one embodiment, said kit further comprises apolymerase. In another embodiment, said kit further comprises amisaminoacylated tRNA. In another embodiment, said kit further comprisesantibodies directed against said epitopes.

Another aspect of the present invention contemplates a method ofintroducing coding sequence for one or more epitope markers into nucleicacid, comprising: a) providing: a first oligonucleotide primercomprising a 5′ portion, a middle portion contiguous with said 5′portion, and a 3′ portion contiguous with said middle portion,wherein 1) said 5′ portion comprises a sequence corresponding to apromoter, 2) said middle portion comprises a sequence corresponding to aribosome binding site, a start codon, and a sequence coding for a firstepitope marker, and 3) said 3′ portion comprises a sequencecomplementary to a first portion of the APC gene (or other diseaserelated gene); ii) a second oligonucleotide primer comprising a 5′portion, a middle portion contiguous with said 5′ portion, and a 3′portion contiguous with said middle portion, wherein 1) said 5′ portioncomprises at least one stop codon, 2) said middle portion comprises asequence encoding for a second epitope marker, and 3) said 3′ portioncomprises a sequence complementary to a second portion of the APC gene(or other disease related gene), wherein either said first epitopemarker or said second epitope marker consist of a portion of the p53amino acid sequence or variant thereof; iii) a polymerase; and iv)template nucleic acid comprising a region of the APC gene (or otherdisease related gene), said region comprising at least said firstportion of the APC gene; and b) mixing said template nucleic acid withsaid first primer, second primer and said polymerase under conditionssuch that amplified template is produced, said amplified templatecomprising said sequence coding for an epitope marker. In oneembodiment, said first and said second oligonucleotide are each lessthan one hundred bases in length. In one embodiment, said sequencecomplementary to a portion of the APC gene of said first and said secondoligonucleotide is 10 bases or greater, but preferably greater than 15bases in length. In another embodiment, said sequence complementary to aportion of the APC gene of said first and said second oligonucleotide isgreater than 20 bases in length. In one embodiment, said firstoligonucleotide has the sequence set forth in SEQ ID NO: 22 and saidsecond oligonucleotide has the sequence set forth in SEQ ID NO: 23. Notintending to limit the present invention, it is understood by oneskilled in the art, that “a region of the APC gene” is larger than “aportion of the APC gene” (just as “regions” of any other gene associatedwith disease are larger than “portions” of the same). For example, aregion of the APC gene may comprise, but is not limited to, the regioncoding for amino acids 1098-1696 (i.e., segment 3).

Another aspect of the present invention contemplates a method,comprising: a) providing: i) an amplified template comprising a firstoligonucleotide having the sequence set forth in SEQ ID NO: 22 and asecond oligonucleotide having the sequence set forth in SEQ ID NO: 23;ii) a misaminoacylated tRNA comprising an affinity marker; and iii) atranslation system; and b) introducing said amplified template and saidmisaminoacylated tRNA into said translation system under conditions suchthat said affinity marker is incorporated into a nascent protein in areaction mixture. In one embodiment, said translation system comprises arabbit reticulocyte lysate. In one embodiment, said affinity markercomprises a biotinyl moiety. In one embodiment, said misaminoacylatedtRNA comprises biotin-lysyl-tRNA. In one embodiment, said methodcomprises after step b), step c) adding a first antibody and a secondantibody to said reaction mixture, or portion thereof, so as to create adiluted reaction mixture. In one embodiment, said first antibodycomprises an antibody reactive with a VSV-derived epitope, said firstantibody conjugated to a first enzyme. In one embodiment, said secondantibody comprises an antibody reactive with a p53-derived epitope, saidsecond antibody conjugated to a second enzyme. In one embodiment, saidmethod further comprises step d) immobilizing said nascent protein bycontacting said nascent protein with a ligand which binds biotin,wherein said ligand is attached to a solid support. In one embodiment,said ligand is selected from the group consisting of avidin andstrepavidin, and variants, mutants and derivatives thereof. In oneembodiment, said misaminoacylated tRNA is a plurality ofmisaminoacylated tRNAs, wherein said plurality of misaminoacylated tRNAsis capable of incorporating said affinity marker at a plurality of aminoacid residues. In one embodiment, said affinity marker comprises afluorescent label.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description, or may be learned from the practice of theinvention.

Definitions

To facilitate understanding of the invention, a number of terms aredefined below.

The term “portion” may refer to a relatively small segment of a proteinor an oligonucleotide. Specifically, a portion of a protein refers to arange of between 5-100 contiguous amino acids while a portion of anucleic acid refers to a range of between 15-300 contiguous nucleicacids.

The term “region” may refer to a relatively large segment of a proteinor an oligonucleotide. Specifically, a region of a protein refers to arange of between 101-1700 contiguous amino acids which a region of anoligonucleotides refers to a range of between 303-5100 contiguousnucleic acids.

The term “contiguous” refers to a continuous, finite, sequence of unitswherein each unit has physical contact with at least one other unit inthe sequence. For example, a contiguous sequence of amino acids arephysically connected by peptide bonds and a contiguous sequence ofnucleic acids are physically connect by phosphodiester bonds.

The term “sequence corresponding to a promoter” refers to a non-codingnucleic acid region that is responsible for the regulation oftranscription (an open reading frame) of the DNA coding for the proteinof interest.

The term “sequence corresponding to a ribosome binding site” refers to acoding nucleic acid region that, when transcribed, allows the binding amRNA in such a manner that translation occurs.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence so long as the desiredenzymatic activity is retained.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three, and usually more than ten. The exact sizewill depend on many factors, which in turn depends on the ultimatefunction or use of the oligonucleotide. The oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may have 5′ and 3′ ends.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

A primer is selected to have on its 3′ end a region that is“substantially” complementary to a strand of specific sequence of thetemplate. A primer must be sufficiently complementary to hybridize witha template strand for primer elongation to occur. A primer sequence neednot reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingsubstantially complementary to the strand. Non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thetemplate to hybridize and thereby form a template primer complex forsynthesis of the extension product of the primer.

As used herein, the terms “hybridize” and “hybridization” refers to theannealing of a complementary sequence to the target nucleic acid. Theability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. Marmur and Lane, Proc. Natl. Acad. Sci.USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461(1960). The terms “annealed” and “hybridized” are used interchangeablythroughout, and are intended to encompass any specific and reproducibleinteraction between an oligonucleotide and a target nucleic acid,including binding of regions having only partial complementarity.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” The term “complement” or“complementary” does not imply or limit pairing to the sense strand orthe antisense strand of a gene; the term is intended to be broad enoughto encompass either situation. Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

The stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m).” The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which on average halfof the base pairs have disassociated.

The term “probe” as used herein refers to an oligonucleotide which formsa duplex structure or other complex with a sequence in another nucleicacid, due to complementarity or other means of reproducible attractiveinteraction, of at least one sequence in the probe with a sequence inthe other nucleic acid.

“Oligonucleotide primers matching or complementary to a gene sequence”refers to oligonucleotide primers capable of facilitating thetemplate-dependent synthesis of single or double-stranded nucleic acids.Oligonucleotide primers matching or complementary to a gene sequence maybe used in PCRs, RT-PCRs and the like. As noted above, anoligonucleotide primer need not be perfectly complementary to a targetor template sequence. A primer need only have a sufficient interactionwith the template that it can be extended by template-dependentsynthesis.

As used herein, the term “poly-histidine tract” or (HIS-tag) refers tothe presence of two to ten histidine residues at either the amino- orcarboxy-terminus of a nascent protein A poly-histidine tract of six toten residues is preferred. The poly-histidine tract is also definedfunctionally as being a number of consecutive histidine residues addedto the protein of interest which allows the affinity purification of theresulting protein on a nickel-chelate column, or the identification of aprotein terminus through the interaction with another molecule (e.g. anantibody reactive with the HIS-tag).

As used herein, the term “marker” is used broadly to encompass a varietyof types of molecules (e.g. introduced into proteins using methods andcompositions of the present invention) which are detectable throughspectral properties (e.g. fluorescent markers) or through functionalproperties (e.g. affinity markers). An epitope marker or “epitope tag”is a marker of the latter type, functioning as a binding site forantibody or other types of binding molecules (e.g. receptors, lectinsand other ligands). Of course, if the epitope marker is used toimmobilize the nascent protein, the epitope marker is also an affinitymarker.

As used herein, the term “total tRNA” is used to describe a mixturecomprising misaminoacylated marker tRNA molecules representing eachamino acid. This mixture has a distinct advantage over the limitedability of misaminoacylated lys-tRNA to reliably incorporate in largevariety of proteins. It is contemplated that “total tRNA” will provide ahomogenous insertion of affinity markers in all nascent proteins.

As used herein, the term “VSV-derived epitope” refers to any amino acidsequence comprising the wild type sequence (i.e., SEQ ID NO:39) ormutations thereof, wherein said mutations include, but are not limitedto, site-specific mutations, deletions, additions, substitutions andtruncations.

As used herein, the term “p53-derived epitope” refers to any amino acidsequence comprising the wild type sequence (i.e., SEQ ID NO:24) ormutations thereof, wherein said mutations include, but are not limitedto, site-specific mutations, frameshift mutations, deletions, additions,substitutions and truncations.

As used herein, the term “VSV variant” refers to any amino acid sequencethat differs from the wild type sequence (i.e., SEQ ID NO: 39) in atleast one, but not more than three residues.

As used herein, the term “p53 variant” refers to any amino acid sequencethat differs from the wild type sequence (i.e., SEQ ID NO: 24) in atleast one, but not more than three residues.

DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows the structure of (A) an amino acid and (B) a peptide (SEQNO:81).

FIG. 2 (SEQ ID NOS:82 to 84) provides a description of the molecularsteps that occur during protein synthesis in a cellular or cell-freesystem.

FIG. 3 shows a structure of (A) a tRNA molecule and (B) approachesinvolved in the aminoacylation of tRNAs.

FIG. 4 is a schematic representation of the method of detecting nascentproteins using fluorescent marker amino acids.

FIG. 5 shows schemes for synthesis and misaminoacylation to tRNA of twodifferent marker amino acids, dansyllysine (scheme 1) and coumarin(scheme 2), with fluorescent properties suitable for the detection ofnascent proteins using gel electrophoresis and UV illumination.

FIG. 6(A) shows chemical compounds containing the 2-nitrobenzyl moiety,and FIG. 6(B) shows cleavage of substrate from a nitrobenzyl linkage.

FIG. 7 provides examples of photocleavable markers.

FIG. 8(A) shows chemical variations of PCB, and FIG. 8(B) depictspossible amino acid linkages.

FIG. 9 shows the photolysis of PCB.

FIG. 10 is a schematic representation of the method for monitoring theproduction of nascent proteins in a cell-free protein expression systemswithout separating the proteins.

FIG. 11 provides examples of non-native amino acids with reporterproperties, illustrates participation of a reporter in proteinsynthesis, and illustrates synthesis of a reporter.

FIG. 12 shows structural components of photocleavable biotin.

FIG. 13 is a schematic representation of the method for introduction ofmarkers at the N-termini of nascent proteins.

FIG. 14 provides a description of the method of detection and isolationof marker in nascent proteins.

FIG. 15 shows the steps in one embodiment for the synthesis ofPCB-lysine.

FIG. 16 provides an experimental strategy for the misaminoacylation oftRNA.

FIG. 17 illustrates dinucleotide synthesis including (i) deoxycytidineprotection, (ii) adenosine protection, and (iii) dinucleotide synthesis.

FIG. 18 depicts aminoacylation of a dinucleotide using marker aminoacids.

FIG. 19 shows the structure of dipyrrometheneborondifluoride(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)dyes.

FIG. 20 is a photograph of a gel showing the incorporation of variousfluorescent molecules into hemolysin during translation.

FIG. 21 shows the incorporation of BODIPY-FL into various proteins. FIG.21A shows the results visualized using laser based Molecular DynamicsFluorImager 595, while FIG. 21B shows the results visualized using aUV-transilluminator.

FIG. 22A shows a time course of fluorescence labeling. FIG. 22B showsthe SDS-PAGE results of various aliquots of the translation mixture,demonstrating the sensitivity of the system.

FIG. 23A is a bar graph showing gel-free quantitation of an N-terminalmarker introduced into a nascent protein in accordance with the methodof the present invention. FIG. 23B is a bar graph showing gel-freequantitation of an C-terminal marker of a nascent protein quantitated inaccordance with the method of the present invention.

FIG. 24 shows gel results for protease treated and untreated protein.

FIG. 25 shows gel results for protein treated with RBCs and untreatedprotein.

FIG. 26A is a gel showing the incorporation of various fluorescentmolecules into α-hemolysin in E. coli translation system usingmisaminoacylated lysyl-tRNA^(lys).

FIG. 26B shows the incorporation of various fluorescent molecules intoluciferase in a TnT wheat germ system using misaminoacylatedlysyl-tRNA^(lys).

FIG. 27 shows gel results of in vitro translation of α-HL carried out inthe presence of various fluorescent-tRNAs, including a tRNA-coumarinderivative.

FIGS. 28A and 28B show mobility shift results by capillaryelectrophoresis.

FIG. 29 are gel results of in vitro translation results wherein threemarkers were introduced into a nascent protein.

FIG. 30 shows Western blot analysis of in vitro translatedtriple-epitope-tagged wild-type p53 (RT-PCR derived DNA). FIG. 30A showsthe total protein staining. FIG. 30B presents the Western blot analysis.

FIG. 31 shows the results for a gel-based PTT of APC Exon 15, segment 2.

FIG. 32 shows the detection of in vitro translated BODIPY-labeledproteins by Western blotting. FIG. 32A shows the results by fluorescenceimaging and FIG. 32B shows the results by Western blotting.

FIG. 33 shows three bar graphs representing the results of a gel-freechemiluminescent protein truncation assay of p53 and APC. FIG. 33A showsthe results for p53 produced by in vitro translation, where the productis captured in a 96-well ELISA plate format using a mouse monoclonalantibody directed against the N-terminal FLAG epitope. FIG. 33B showsthe results for p53 produced by in vitro translation, where the productis captured in a 96-well ELISA plate format using a nickel chelateplate. FIG. 33C shows the results for APC produced by in vitrotranslation, where the product is captured on nickel metal chelate96-well ELISA plates. All WT N- and C-terminal signals as well as mutantN-terminal signals were normalized to 100%.

FIG. 34 is a gel showing immunoprecipitation results for p53 andHemolysin.

FIG. 35 schematically shows a synthesis scheme for the preparation ofBODIPY-Val-pdCpA.

FIG. 36 schematically shows a synthesis scheme for the attachment of theBODIPY-Val-pdCpA conjugate of FIG. 35 to tRNA.

FIG. 37 shows the result of translation reactions using the charged tRNAof FIG. 36.

FIG. 38 schematically shows one embodiment of a drug/proteome screeningassay.

FIG. 39 schematically shows one embodiment of a drug/proteome screeningassay.

FIG. 40 is a bar graph showing the results of a drug/proteome screeningassay.

FIG. 41 depicts a schematic representation of one embodiment of aHigh-Throughput Solid-phase Protein Truncation Test (HTS-PTT).

FIG. 42 displays ELISA-based HTS-PTT data for mutations in the APCsegment 3 gene. Four FAP patients (P1-P4) are compared to cell-linehomozygous mutations (C₁-C₃) and assay normalization of 100 based ondata from wild-type (WT) DNA. Selective capture occurred on NeutrAvidin™plates via biotin incorporated by biotin-lysyl-tRNA. Epitope tags weredetected by using either antibody-conjugated horseradish peroxidase(HRP) or alkaline phosphatase (AP). All data points represent andaverage of ≧3 replicates and error bars indicate the standard deviation.

FIG. 43 shows a gel-based PTT assay with fluorescent readout performedon 4 μl aliquots of samples comprising the data of FIG. 42. BL1:controls without tRNAs and DNA. BL2: controls without DNA. Asterisk:Auto-fluorescent control protein marker band. The mutations confirmed byDNA sequencing are: C1: Q1367→Stop; C2: ΔC at codon 1416; C3:Q1338→Stop; P1: ΔAG at codon 1536; P2: ΔC at codon 1200; P3: ΔC at codon1536 and P4: Insertion of A at codon 1555. All mutations are found inthe APC mutation database.

FIG. 44 displays HTS-PTT truncation mutation detection in APC gene atvarious dilutions of WT:C3 DNA (circles-solid line) or mRNA(triangles-broken line). The DNA were mixed prior to PCR. The mRNA weremixed prior to isolation. All data points represent an average of ≧3replicates and error bars indicate the standard deviation.

FIG. 45 displays the same data as in FIG. 44 using a color coded matrixarray. C/N terminal ratios were reproduced by using MICROSOFT POWERPOINTXP software to mix Green (g) and Red (r) RGB values. Blue was set at 0while Red and Green ranged from 0-255. Color adjustments were made basedon R value and C/N terminal ratio by using the formulas; r=255 (R/100)and g=255 [1−(R/100)].

FIG. 46 displays data comparing the signals between biotin-tRNA^(lys)(grey bars) and biotin-tRNA^(TOTAL) (black bars) for the VSV epitope orthe P53 epitope. WT: Wild-type DNA. F3: Human Truncated Mutant DNA K3:Human Truncated Mutant DNA. The differences in translation efficiency isreflected in the respective signal intensities between the VSV and P53markers.

FIG. 47 displays data comparing the signals between standardincorporation of biotin-tRNA^(lys) on an unmodified nascent protein(grey bars) and PCR insertion of five (5) extra terminal lysine residueson a nascent protein (black bars). WT: Wild-Type DNA. N3: HumanTruncated Mutant DNA.

FIG. 48 depicts the separation by SDS-PAGE of APC-23 DNA (140 kD) fromAPC-3 DNA (70 kD).

FIG. 49 compares HTS-PTT evaluation of APC-3 DNA (grey bars) and APC-23DNA (black bars) in WT: Wild type DNA. A3: Homozygous Mutant Human DNA.F3: Homozygotic Mutant Human DNA. K3: Heterozygous Mutant DNA. N3:Heterozygotic Mutant Human DNA.

FIG. 50 shows separation of PCR APC-3 product corresponding to a singleDNA copy following amplification with a very small amount of DNA.

FIG. 51 displays C-Terminal fluorescence signal using MicroArray PTT.BL: Negative control. 0, 50, 75 and 100 represent percentage of WT DNA.

FIG. 52 illustrates High Sensitivity Chemiluminescent “Gel-Free” PTT fornon-invasive colorectal cancer screening.

FIG. 53 demonstrates the detection of truncation mutations via selectiveincorporation of photocleavable (PC)-Biotin. Panel A shows signalsobtained for mixtures containing 2%, 1% and 0.4% mixtures ofWild-Type:Mutant DNA of α-hemolysin. Panel B shows signals obtained formixtures containing 5% and 20% DNA mixtures.

FIG. 54 shows the data of FIG. 53 replotted into a regression curve.

FIG. 55 demonstrates High Sensitivity Gel-Based Fluorescent PTT analysisof APC segment 2 (APC-2) for non-invasive colorectal cancer screening.Panel A: 0%, 50%, 25%, 10%, 4% and 2% represent mixtures of Wild-Type tomutant DNA. −DNA: Negative control without DNA. M: 100% mutant DNA.Panel B: Regression curve calculated from densitometry data.

FIG. 56 shows an expected elution pattern using capillaryelectrophoresis.

FIG. 57 displays the detection of a point mutation in in vitro expressedα-hemolysin by MALDI-TOF. Tracing a: 34,884-WT singly ionized species;[MH]²⁺-WT doubly ionized species. Tracing b: 34,982-mutant singlyionized species; [MH]²⁺-mutant doubly ionized species.

FIG. 58 compares the signal strength of equal amounts of VSV sandwichantibody detection (grey bars) with VSV-HRP directly conjugated antibodydetection (black bars) in 100% WT, 90% WT:10% Mutant, 75% WT:25% Mutant,50% WT:50% Mutant, and 100% Mutant samples.

FIG. 59 compares the 2-Step ELISA (grey bars) with the 1-Step ELISA(black bars).

FIG. 60 depicts C/N ratios after separate antibody incubation (greybars) or mixed antibody incubation (black bars).

FIG. 61 demonstrates the high sensitivity of ELISA-PTT. C-terminal andN-terminal data from samples containing from between 0-2 μl oftranslation mix are shown.

FIG. 62 illustrates the application of ELISA to rapid screening of cDNAlibrary expression.

FIG. 63 provides data showing a time course of protease substratedegradation monitored by ELISA.

FIG. 64 provides data showing a time course of product appearance duringcaspase-3 digestion using C-terminal tags monitored by ELISA.

FIG. 65 exemplifies the use of ELISA in monitoring plasmid expression byenzyme markers.

FIG. 66 shows an schematic for an E. coli initiator tRNA comprising afluorescent reporter group such as 4-thio-uridine (black dot).

FIG. 67 shows one possible tertiary structure for an E. coli initiatortRNA.

FIG. 68 provides several exemplary quencher-fluorescent pairsidentifying their respective R₀ values

FIG. 69 shows the structure of QSY® 7 maleimide (Q-10257) exemplifyingone embodiment of a fluorescent reporter.

FIG. 70 displays the amino acid sequence for the full-length wild-typecellular tumor antigen p53 (Accession No.: DNHU53) (SEQ ID NO:49).

FIG. 71 displays the nucleic acid sequence of the human phosphoproteinp53 gene exon 11 encoding the full length wild-type cellular tumorantigen p53 (Accession No.: M13121 N00032) (SEQ ID NO:50).

DESCRIPTION OF THE INVENTION

As embodied and described herein, the present invention comprisesmethods for the non-radioactive labeling and detection of the productsof new or nascent protein synthesis, and methods for the isolation ofthese nascent proteins from preexisting proteins in a cellular orcell-free translation system. As radioactive labels are not used, thereare no special measures which must be taken to dispose of wastematerials. There is also no radioactivity danger or risk which wouldprevent further utilization of the translation product as occurs whenusing radioactive labels and the resulting protein product may be useddirectly or further purified. In addition, no prior knowledge of theprotein sequence or structure is required which would involve, forexample, unique suppressor tRNAs. Further, the sequence of the gene ormRNA need not be determined. Consequently, the existence of non-sensecodons or any specific codons in the coding region of the mRNA is notnecessary. Any tRNA can be used, including specific tRNAs for directedlabeling, but such specificity is not required. Unlikepost-translational labeling, nascent proteins are labeled withspecificity and without being subjected to post-translationalmodifications which may effect protein structure or function.

One embodiment of the invention is directed to a method for labelingnascent proteins synthesized in a translation system. These proteins arelabeled while being synthesized with detectable markers which areincorporated into the peptide chain. Markers which are aminoacylated totRNA molecules, may comprise native amino acids, non-native amino acids,amino acid analogs or derivatives, or chemical moieties. These markersare introduced into nascent proteins from the resulting misaminoacylatedtRNAs during the translation process. Aminoacylation is the processwhereby a tRNA molecule becomes charged. When this process occurs invivo, it is referred to as natural aminoacylation and the resultingproduct is an aminoacylated tRNA charged with a native amino acid. Whenthis process occurs through artificial means, it is calledmisaminoacylation and a tRNA charged with anything but a native aminoacid molecule is referred to as a misaminoacylated tRNA.

According to the present method, misaminoacylated tRNAs are introducedinto a cellular or cell-free protein synthesizing system, thetranslation system, where they function in protein synthesis toincorporate detectable marker in place of a native amino acid in thegrowing peptide chain. The translation system comprises macromoleculesincluding RNA and enzymes, translation, initiation and elongationfactors, and chemical reagents. RNA of the system is required in threemolecular forms, ribosomal RNA (rRNA), messenger RNA (mRNA) and transferRNA (tRNA). mRNA carries the genetic instructions for building a peptideencoded within its codon sequence. tRNAs contain specific anti-codonswhich decode the mRNA and individually carry amino acids into positionalong the growing peptide chain. Ribosomes, complexes of rRNA andprotein, provide a dynamic structural framework on which the translationprocess, including translocation, can proceed. Within the cell,individualized aminoacyl tRNA synthetases bind specific amino acids totRNA molecules carrying the matching anti-codon creating aminoacylatedor charged tRNAs by the process of aminoacylation. The process oftranslation including the aminoacylation or charging of a tRNA moleculeis described in Molecular Cell Biology (J. Darnell et al. editors,Scientific American Books, N.Y., N.Y. 1991), which is herebyspecifically incorporated by reference. Aminoacylation may be natural orby artificial means utilizing native amino acids, non-native amino acid,amino acid analogs or derivatives, or other molecules such as detectablechemicals or coupling agents. The resulting misaminoacylated tRNAcomprises a native amino acid coupled with a chemical moiety, non-nativeamino acid, amino acid derivative or analog, or other detectablechemicals. These misaminoacylated tRNAs incorporate their markers intothe growing peptide chain during translation forming labeled nascentproteins which can be detected and isolated by the presence or absenceof the marker.

Any proteins that can be expressed by translation in a cellular orcell-free translation system may be nascent proteins and consequently,labeled, detected and isolated by the methods of the invention. Examplesof such proteins include enzymes such as proteolytic proteins,cytokines, hormones, immunogenic proteins, carbohydrate or lipid bindingproteins, nucleic acid binding proteins, human proteins, viral proteins,bacterial proteins, parasitic proteins and fragments and combinations.These methods are well adapted for the detection of products ofrecombinant genes and gene fusion products because recombinant vectorscarrying such genes generally carry strong promoters which transcribemRNAs at fairly high levels. These mRNAs are easily translated in atranslation system.

Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include whole cellpreparations such as permeabilized cells or cell cultures wherein adesired nucleic acid sequence can be transcribed to mRNA and the mRNAtranslated.

Cell-free translation systems are commercially available and manydifferent types and systems are well-known. Examples of cell-freesystems include prokaryotic lysates such as Escherichia coli lysates,and eukaryotic lysates such as wheat germ extracts, insect cell lysates,rabbit reticulocyte lysates, frog oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified becausemany such modifications are only possible in eukaryotic systems. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, such as the caninepancreatic extracts containing microsomal membranes, are also availablewhich are useful for translating secretory proteins. Mixtures ofpurified translation factors have also been used successfully totranslate mRNA into protein as well as combinations of lysates orlysates supplemented with purified translation factors such asinitiation factor-1 (IF-1), IF-2, IF-3 (α or β), elongation factor T(EF-Tu), or termination factors.

Cell-free systems may also be coupled transcription/translation systemswherein DNA is introduced to the system, transcribed into mRNA and themRNA translated as described in Current Protocols in Molecular Biology(F. M. Ausubel et al. editors, Wiley Interscience, 1993), which ishereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

tRNA molecules chosen for misaminoacylation with marker do notnecessarily possess any special properties other than the ability tofunction in the protein synthesis system. Due to the universality of theprotein translation system in living systems, a large number of tRNAscan be used with both cellular and cell-free reaction mixtures. SpecifictRNA molecules which recognize unique codons, such as nonsense or ambercodons (UAG), are not required.

Site-directed incorporation of the normative analogs into the proteinduring translation is also not required. Incorporation of markers canoccur anywhere in the polypeptide and can also occur at multiplelocations. This eliminates the need for prior information about thegenetic sequence of the translated mRNA or the need for modifying thisgenetic sequence.

In some cases, it may be desirable to preserve the functional propertiesof the nascent protein. A subset of tRNAs which will incorporate markersat sites which do not interfere with protein function or structure canbe chosen. Amino acids at the amino or carboxyl terminus of apolypeptide do not alter significantly the function or structure. tRNAmolecules which recognize the universal codon for the initiation ofprotein translation (AUG), when misaminoacylated with marker, will placemarker at the amino terminus. Prokaryotic protein synthesizing systemsutilize initiator tRNA^(fMet) molecules and eukaryotic systems initiatortRNA^(Met) molecules. In either system, the initiator tRNA molecules areaminoacylated with markers which may be non-native amino acids or aminoacid analogs or derivatives that possess marker, reporter or affinityproperties. The resulting nascent proteins will be exclusively labeledat their amino terminus, although markers placed internally do notnecessarily destroy structural or functional aspects of a protein. Forexample, a tRNA^(LYS) may be misaminoacylated with the amino acidderivative dansyllysine which does not interfere with protein functionor structure. In addition, using limiting amounts of misaminoacylatedtRNAs, it is possible to detect and isolate nascent proteins having onlya very small fraction labeled with marker which can be very useful forisolating proteins when the effects of large quantities of marker wouldbe detrimental or are unknown.

tRNAs molecules used for aminoacylation are commercially available froma number of sources and can be prepared using well-known methods fromsources including Escherichia coli, yeast, calf liver and wheat germcells (Sigma Chemical; St. Louis, Mo.; Promega; Madison, Wis.;Boehringer Mannheim Biochemicals; Indianapolis, Ind.). Their isolationand purification mainly involves cell-lysis, phenol extraction followedby chromatography on DEAE-cellulose. Amino-acid specific tRNA, forexample tRNA^(fMet), can be isolated by expression from cloned genes andoverexpressed in host cells and separated from total tRNA by techniquessuch as preparative polyacrylamide gel electrophoresis followed by bandexcision and elution in high yield and purity (Seong and RajBhandary,Proc. Natl. Acad. Sci. USA 84:334-338′, 1987). Run-off transcriptionallows for the production of any specific tRNA in high purity, but itsapplications can be limited due to lack of post-transcriptionalmodifications (Bruce and Uhlenbeck, Biochemistry 21:3921, 1982).

Misaminoacylated tRNAs are introduced into the cellular-or cell-freeprotein synthesis system. In the cell-free protein synthesis system, thereaction mixture contains all the cellular components necessary tosupport protein synthesis including ribosomes, tRNA, rRNA, spermidineand physiological ions such as magnesium and potassium at appropriateconcentrations and an appropriate pH. Reaction mixtures can be normallyderived from a number of different sources including wheat germ, E. coli(S-30), red blood cells (reticulocyte lysate,) and oocytes, and oncecreated can be stored as aliquots at about +4° C. to −70° C. The methodof preparing such reaction mixtures is described by J. M. Pratt(Transcription and Translation, B. D. Hames and S. J. Higgins, Editors,p. 209, IRL Press, Oxford, 1984) which is hereby incorporated byreference. Many different translation systems are commercially availablefrom a number of manufacturers.

The misaminoacylated tRNA is added directly to the reaction mixture as asolution of predetermined volume and concentration. This can be donedirectly prior to storing the reaction mixture at −70° C. in which casethe entire mixture is thawed prior to initiation of protein synthesis orprior to the initiation of protein synthesis. Efficient incorporation ofmarkers into nascent proteins is sensitive to the final pH and magnesiumion concentration. Reaction mixtures are normally about pH 6.8 andcontain a magnesium ion concentration of about 3 mM. These conditionsimpart stability to the base-labile aminoacyl linkage of themisaminoacylated tRNA. Aminoacylated tRNAs are available in sufficientquantities from the translation extract. Misaminoacylated tRNAs chargedwith markers are added at between about 1.0 μg/ml to about 1.0 mg/ml,preferably at between about 10 μg/ml to about 500 μg/ml, and morepreferably at about 150 μg/ml.

Initiation of protein synthesis occurs upon addition of a quantity ofmRNA or DNA to the reaction mixture containing the misaminoacylatedtRNAs. mRNA molecules may be prepared or obtained from recombinantsources, or purified from other cells by procedure such as poly-dTchromatography. One method of assuring that the proper ratio of thereaction mixture components is to use predetermined volumes that arestored in convenient containers such as vials or standardmicrocentrifuge tubes. For example, DNA and/or mRNA coding for thenascent proteins and the misaminoacylated tRNA solution are premixed inproper amounts and stored separately in tubes. Tubes are mixed whenneeded to initiate protein synthesis.

Translations in cell-free systems generally require incubation of theingredients for a period of time. Incubation times range from about 5minutes to many hours, but is preferably between about thirty minutes toabout five hours and more preferably between about one to about threehours. Incubation may also be performed in a continuous manner wherebyreagents are flowed into the system and nascent proteins removed or leftto accumulate using a continuous flow system (A. S. Spirin et al., Sci.242:1162-64, 1988). This process may be desirable for large scaleproduction of nascent proteins. Incubations may also be performed usinga dialysis system where consumable reagents are available for thetranslation system in an outer reservoir which is separated from largercomponents of the translation system by a dialysis membrane [Kim, D.,and Choi, C. (1996) Biotechnol Prog 12, 645-649]. Incubation times varysignificantly with the volume of the translation mix and the temperatureof the incubation. Incubation temperatures can be between about 4° C. toabout 60° C., and are preferably between about 15° C. to about 50° C.,and more preferably between about 25° C. to about 45° C. and even morepreferably at about 25° C. or about 37° C. Certain markers may besensitive to temperature fluctuations and in such cases, it ispreferable to conduct those incubations in the non-sensitive ranges.Translation mixes will typically comprise buffers such as Tris-HCl,Hepes or another suitable buffering agent to maintain the pH of thesolution between about 6 to 8, and preferably at about 7. Again, certainmarkers may be pH sensitive and in such cases, it is preferable toconduct incubations outside of the sensitive ranges for the marker.Translation efficiency may not be optimal, but marker utility will beenhanced. Other reagents which may be in the translation system includedithiothreitol (DTT) or 2-mercaptoethanol as reducing agents, RNasin toinhibit RNA breakdown, and nucleoside triphosphates or creatinephosphate and creatine kinase to provide chemical energy for thetranslation process.

In cellular protein synthesis, it is necessary to introducemisaminoacylated tRNAs or markers into intact cells, cell organelles,cell envelopes and other discrete volumes bounded by an intactbiological membrane, which contain a protein synthesizing system. Thiscan be accomplished through a variety of methods that have beenpreviously established such as sealing the tRNA solution into liposomesor vesicles which have the characteristic that they can be induced tofuse with cells. Fusion introduces the liposome or vesicle interiorsolution containing the tRNA into the cell. Alternatively, some cellswill actively incorporate liposomes into their interior cytoplasmthrough phagocytosis. The tRNA solution could also be introduced throughthe process of cationic detergent mediated lipofection (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-17, 1987), or injected into largecells such as oocytes. Injection may be through direct perfusion withmicropipettes or through the method of electroporation.

Alternatively, cells can be permeabilized by incubation for a shortperiod of time in a solution containing low concentrations of detergentsin a hypotonic media. Useful detergents include Nonidet-P 40 (NP40),Triton X-100 (TX-100) or deoxycholate at concentrations of about 0.01 nMto 1.0 mM, preferably between about 0.1 μM to about 0.01 mM, and morepreferably about 1 μM. Permeabilized cells allow marker to pass throughcellular membranes unaltered and be incorporated into nascent proteinsby host cell enzymes. Such systems can be formed from intact cells inculture such as bacterial cells, primary cells, immortalized cell lines,human cells or mixed cell populations. These cells may, for example, betransfected with an appropriate vector containing the gene of interest,under the control of a strong and possibly regulated promoter. Messagesare expressed from these vectors and subsequently translated withincells. Intact misaminoacylated tRNA molecules, already charged with anon-radioactive marker could be introduced to cells and incorporatedinto translated product.

One example of the use of misaminoacylation to detect nascent protein isschematically represented in FIG. 4. A tRNA molecule is misaminoacylatedwith the marker which is highly fluorescent when excited with UV(ultraviolet) radiation. The misaminoacylated tRNA is then introducedinto a cell-free protein synthesis extract and the nascent proteinscontaining the marker analog produced. Proteins in the cell-free extractare separated by polyacrylamide gel electrophoresis (PAGE). Theresulting gel contains bands which correspond to all of the proteinspresent in the cell-free extract. The nascent protein is identified uponUV illumination of the gel by detection of fluorescence from the bandcorresponding to proteins containing marker. Detection can be throughvisible observation or by other conventional means of fluorescencedetection.

The misaminoacylated tRNA can be formed by natural aminoacylation usingcellular enzymes or misaminoacylation such as chemicalmisaminoacylation. One type of chemical misaminoacylation involvestruncation of the tRNA molecule to permit attachment of the marker ormarker precursor. For example, successive treatments with periodate pluslysine, pH 8.0, and alkaline phosphatase removes 3′-terminal residues ofany tRNA molecule generating tRNA-OH-3′ with a mononucleotide ordinucleotide deletion from the 3′-terminus (Neu and Heppel, J. Biol.Chem. 239:2927-34, 1964). Alternatively, tRNA molecules may begenetically manipulated to delete specific portions of the tRNA gene.The resulting gene is transcribed producing truncated tRNA molecules(Sampson and Uhlenbeck, Proc. Natl. Acad. Sci. USA 85:1033-37, 1988).Next, a dinucleotide is chemically linked to a modified amino acid orother marker by, for example, acylation. Using this procedure, markerscan be synthesized and acylated to dinucleotides in high yield (Hudson,J. Org. Chem. 53:617-624, 1988; Happ et al., J. Org. Chem. 52:5387-91,1987). These modified groups are bound together and linked via thedinucleotide to the truncated tRNA molecules in a process referred to asligase coupling (FIG. 3B).

A different bond is involved in misaminoacylation (FIG. 3B, link B) thanthe bond involved with activation of tRNA by aminoacyl tRNA synthetase(FIG. 3B, link A). As T4 RNA ligase does not recognize the acylsubstituent, tRNA molecules can be readily misaminoacylated with fewchemical complications or side reactions (link B, FIG. 3B) (T. G.Heckler et al., Biochemistry 23:1468-73, 1984; and T. G. Heckler et al.,Tetrahedron 40:87-94, 1984). This process is insensitive to the natureof the attached amino acid and allows for misaminoacylation using avariety of non-native amino acids. In contrast, purely enzymaticaminoacylation (link A) is highly sensitive and specific for thestructures of substrate tRNA and amino acids.

Markers are basically molecules which will be recognized by the enzymesof the translation process and transferred from a charged tRNA into agrowing peptide chain. To be useful, markers must also possess certainphysical and physio-chemical properties. Therefore, there are multiplecriteria which can be used to identify a useful marker. First, a markermust be suitable for incorporation into a growing peptide chain. Thismay be determined by the presence of chemical groups which willparticipate in peptide bond formation. Second, markers should beattachable to a tRNA molecule. Attachment is a covalent interactionbetween the 3′-terminus of the tRNA molecule and the carboxy group ofthe marker or a linking group attached to the marker and to a truncatedtRNA molecule. Linking groups may be nucleotides, short oligonucleotidesor other similar molecules and are preferably dinucleotides and morepreferably the dinucleotide CA. Third, markers should have one or morephysical properties that facilitate detection and possibly isolation ofnascent proteins. Useful physical properties include a characteristicelectromagnetic spectral property such as emission or absorbance,magnetism, electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity.

Useful markers are native amino acids coupled with a detectable label,detectable non-native amino acids, detectable amino acid analogs anddetectable amino acid derivatives. Labels and other detectable moietiesmay be ferromagnetic, paramagnetic, diamagnetic, luminescent,electrochemiluminescent, fluorescent, phosphorescent, chromatic or havea distinctive mass. Fluorescent moieties which are useful as markersinclude dansyl fluorophores, coumarins and coumarin derivatives,fluorescent acridinium moieties and benzopyrene based fluorophores.Preferably, the fluorescent marker has a high quantum yield offluorescence at a wavelength different from native amino acids and morepreferably has high quantum yield of fluorescence can be excited in boththe UV and visible portion of the spectrum. Upon excitation at apreselected wavelength, the marker is detectable at low concentrationseither visually or using conventional fluorescence detection methods.Electrochemiluminescent markers such as ruthenium chelates and itsderivatives or nitroxide amino acids and their derivatives are preferredwhen extreme sensitivity is desired (J. DiCesare et al., BioTechniques15:152-59, 1993). These markers are detectable at the femtomolar rangesand below.

In addition to fluorescent markers, a variety of markers possessingother specific physical properties can be used to detect nascent proteinproduction. In general, these properties are based on the interactionand response of the marker to electromagnetic fields and radiation andinclude absorption in the UV, visible and infrared regions of theelectromagnetic spectrum, presence of chromophores which are Ramanactive, and can be further enhanced by resonance Raman spectroscopy,electron spin resonance activity and nuclear magnetic resonances and useof a mass spectrometer to detect presence of a marker with a specificmolecular mass. These electromagnetic spectroscopic properties arepreferably not possessed by native amino acids or are readilydistinguishable from the properties of native amino acids. For example,the amino acid tryptophan absorbs near 290 nm, and has fluorescentemission near 340 nm when excited with light near 290 nm. Thus,tryptophan analogs with absorption and/or fluorescence properties thatare sufficiently different from tryptophan can be used to facilitatetheir detection in proteins.

Many different modified amino acids which can be used as markers arecommercially available (Sigma Chemical; St. Louis, Mo.; MolecularProbes; Eugene, Oreg.). One such marker is Nε-dansyllysine created bythe misaminoacylation of a dansyl fluorophore to a tRNA molecule (FIG.5; scheme 1). The α-amino group of Nε-dansyllysine is first blocked withNVOC (ortho-nitro veratryl oxycarbonyl chloride) and the carboxyl groupactivated with cyanomethyl ester. Misaminoacylation is performed asdescribed. The misaminoacylated tRNA molecules are then introduced intothe protein synthesis system, whereupon the dansyllysine is incorporateddirectly into the newly synthesized proteins.

Another such marker is a fluorescent amino acid analog based on thehighly fluorescent molecule coumarin (FIG. 5; scheme 2). Thisfluorophore has a much higher fluorescence quantum yield than dansylchloride and can facilitate detection of much lower levels of nascentprotein. In addition, this coumarin derivative has a structure similarto the native amino acid tryptophan. These structural similarities areuseful where maintenance of the nascent proteins' native structure orfunction are important or desired. Coumarin is synthesized as depictedin FIG. 5 (scheme 2). Acetamidomalonate is alkylated with a slightexcess of 4-bromomethyl coumarin (Aldrich Chemicals; Milwaukee; Wis.) inthe presence of sodium ethoxide followed by acid hydrolysis. Thecorresponding amino acid as a hydrochloride salt that can be convertedto the free amino acid analog.

The coumarin derivative can be used most advantageously if itmisamino-acylates the tryptophan-tRNA, either enzymatically orchemically. When introduced in the form of the misaminoacylatedtryptophan-tRNA, the coumarin amino acid will be incorporated only intotryptophan positions. By controlling the concentration ofmisaminoacylated tRNAs or free coumarin derivatives in the cell-freesynthesis system, the number of coumarin amino acids incorporated intothe nascent protein can also be controlled. This procedure can beutilized to control the amount of most any markers in nascent proteins.

Markers can be chemically synthesized from a native amino acid and amolecule with marker properties which cannot normally function as anamino acid. For example a highly fluorescent molecule can be chemicallylinked to a native amino acid group. The chemical modification can occuron the amino acid side-chain, leaving the carboxyl and aminofunctionalities free to participate in a polypeptide bond formation.Highly fluorescent molecules (e.g. dansyl chloride) can be linked to thenucleophilic side chains of a variety of amino acids including lysine,arginine, tyrosine, cysteine, histidine, etc., mainly as a sulfonamidefor amino groups or sulfate bonds to yield fluorescent derivatives. Suchderivatization leaves the ability to form peptide bond intact, allowingthe normal incorporation of dansyllysine into a protein.

A number of factors determine the usefulness of a marker which is to beincorporated into nascent proteins through misaminoacylated tRNAs. Theseinclude the ability to incorporate the marker group into the proteinthrough the use of a misaminoacylated tRNA in a cell-free or cellularprotein synthesis system and the intrinsic detectability of the markeronce it is incorporated into the nascent protein. In general, markerswith superior properties will allow shorter incubation times and requiresmaller samples for the accurate detection of the nascent proteins.These factors directly influence the usefulness of the methodsdescribed. In the case of fluorescent markers used for the incorporationinto nascent proteins, favorable properties can be but are not limitedto, small size, high quantum yield of fluorescence, and stability toprolonged light exposure (bleach resistance).

Even with knowledge of the above factors, the optimum conditions toincorporate a specific marker into a protein using a specific cell-freeor cellular translation system is difficult to predict a priori since itdepends on the detailed interaction of the marker group with componentsof the protein translational synthesis system including the tRNA,initiation or elongation factors and components of the ribosome. Whileit is generally expected that markers with smaller sizes can beaccommodated more readily into the ribosome, the exact shape of themolecule and its specific interactions in the ribosomal binding sitewill be the most important determinant. For this reason, it is possiblethat some markers which are larger in size can be more readilyincorporated into nascent proteins compared to smaller markers. Forexample, such factors are very difficult to predict using known methodsof molecular modeling.

One group of fluorophores with members possessing several favorableproperties (including favorable interactions with components of theprotein translational synthesis system) is the group derived fromdipyrrometheneboron difluoride derivatives (BODIPY) (FIG. 19). Comparedto a variety of other commonly used fluorophores with advantageousproperties such as high quantum yields, some BODIPY compounds have theadditional unusual property that they are highly compatible with theprotein synthesis system. The core structure of all BODIPY fluorophoresis 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. See U.S. Pat. Nos.4,774,339; 5,187,288; 5,248,782; 5,274,113; 5,433,896; 5,451,663, allhereby incorporated by reference. A central feature is a difluoroboronas shown in FIG. 19. All BODIPY fluorophores have several desirableproperties for a marker (Molecular Probes Catalog, pages 13-18)including a high extinction coefficient, high fluorescence quantumyield, spectra that are insensitive to solvent polarity and pH, narrowemission bandwidth resulting in a higher peak intensity compared toother dyes such as fluorescein, absence of ionic charge and enhancedphotostability compared to fluorescein. The addition of substituents tothe basic BODIPY structure which cause additional conjugation can beused to shift the wavelength of excitation or emission to convenientwavelengths compatible with the means of detection.

These dyes were described for the first time by Vos de Waal et al.(1977) and its fluorescence properties subsequently described by Wories[See Wories et al., “A novel water-soluble fluorescent probe: Synthesis,luminescence and biological properties of the sodium salt of the4-sulfonato-3,3′, 5,5′-tetramethyl-2,2′-pyrromethen-1,1′-BF.sub.2complex,” Recl. Trav. Chim. PAYSBAS 104, 288 (1985). Dyes derived fromdipyrrometheneboron difluoride have additional characteristics that makethem suitable for incorporation into nascent proteins. Simple alkylderivatives of the fluorophore4,4-difluoro-4-bora-3a,4a-diaza-s-indacene have been described by Treibs& Kreuzer, [Difluorboryl-komplexe von di-und tripyrrylmethenen, LIEBIGSANNALEN CHEM. 718, 208 (1968)] and by Worries, Kopek, Lodder, &Lugtenburg, [A novel water-soluble fluorescent probe: Synthesis,luminescence and biological properties of the sodium salt of the4-sulfonato-3,3′,5,5′-tetramethyl-2,2′-pyrromethen-1,1′-BF.sub.2complex, RECL. TRAV. CHIM. PAYS-BAS 104, 288 (1985)] as being highlyfluorescent with spectral properties that are similar to fluorescein,with maximum absorbance at about 490 to 510 nm and maximum emission atabout 500 to 530 nm. U.S. Pat. No. 4,774,339 to Haugland et al. (1988)('339 patent) (hereby incorporated by reference) describes4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (dipyrrometheneborondifluoride)dyes including hydrogen, halogen, alkyl, cycloalkyl, aryl,arylalkyl, acyl, and sulfo-substituted derivatives that contain reactivegroups suitable for conjugation to biomolecules, that have goodphotostability, and which have fluorescein-like spectra. As described inthe '339 patent, and by Pavlopoulos, et al., [Laser action from atetramethylpyrromethene-BF.sub.2 complex, APP. OPTICS 27, 4998 (1988)],the emission of the alkyl derivatives of4,4-difluoro-4-bora-3a,4a-diaza-s-indacene fluorescent dyes clearlyoverlaps that of fluorescein. The overlap allows the alkyl derivativesof dipyrrometheneboron difluoride to be used with the same opticalequipment as used with fluorescein-based dyes without modification ofthe excitation sources or optical filters. Similarly, aryl/heteroarylsubstituents in the dipyrrometheneboron difluoride cause the maximum ofabsorbance/emission to shift into longer wavelengths (See U.S. Pat. No.5,451,663 hereby incorporated by reference).

A variety of BODIPY molecules are commercially available in an aminereactive form which can be used to derivatize aminoacylated tRNAs toyield a misaminoacylated tRNA with a BODIPY marker moiety. One exampleof a compound from this family which exhibits superior properties forincorporation of a detectable marker into nascent proteins is4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY-FL).When the sulfonated N-hydroxysuccinimide (NHS) derivative of BODIPY-FLis used to misaminoacylate an E. coli initiator tRNA^(fmet), the nascentprotein produced can be easily detected on polyacyrlamide gels afterelectrophoresis using a standard UV-transilluminator and photographic orCCD imaging system. This can be accomplished by using purifiedtRNA^(fmet) which is first aminoacylated with methionine and then theα-amino group of methionine is specifically modified usingN-hydroxysuccinimide BODIPY. Before the modification reaction, thetRNA^(fmet) is charged maximally (>90%) and confirmed by using³⁵S-methionine and acid-urea gels [Varshney, U., Lee, C. P., andRajBhandary, U. L. 1991. Direct analysis of aminoacylation levels oftRNA in vitro. J. Biol. Chem. 266:24712-24718].

Less than 10 nanoliters of a commercially available E. coli extract (E.coli T7 translation system, Promega, Madison, Wis.) are needed foranalysis corresponding to less than 1 ng of synthesized protein.Incubation times required to produce detectable protein is approximately1 hour but can be as little as 5 minutes (or less). BODIPY-FL can alsobe detected with higher sensitivity using commercially availablefluorescent scanners with 488 nm excitation and emission measurementabove 520 nm. Similar tests using other commercially available dyesincluding NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), and Pyrine-PyMPO showapproximately an order of magnitude reduction in fluorescence makingthem more difficult to detect using standard laboratory equipment suchas a UV-transilluminator or fluorescent scanner. It has previously beenshown that fluorescent markers such as3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3,-diaminoproprionic acid(NBD-DAP) and coumarin could be incorporated into proteins usingmisaminoacylated tRNAs. However, detection of nascent proteinscontaining these markers was only demonstrated using highly sensitiveinstrumentation such a fluorescent spectrometer or amicrospectrofluorometer and often require indirect methods such as theuse of fluorescence resonance energy transfer (FRET) (Turcatti, G.,Nemeth, K., Edgerton, M. D., Meseth, U., Talabot, F., Peitsch, M.,Knowles, J., Vogel, H., and Chollet, A. (1996) J Biol Chem 271(33),19991-8; Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, B. (1994)J Mol Biol 244(3), 319-31). Such instruments are generally not availablefor routine use in a molecular biology laboratory and only with specialadaptation can be equipped for measurement of fluorescent bands on agel.

An additional advantage of BODIPY-FL as a marker is the availability ofmonoclonal antibodies directed against it which can be used to affinitypurify nascent proteins containing said marker. One example of such amonoclonal antibody is anti-BODIPY-FL antibody (Cat# A-5770, MolecularProbes, Eugene, Oreg.). This combined with the ability incorporateBODIPY-FL into nascent proteins with high efficiency relative to othercommercially available markers using misaminoacylated tRNAs facilitatesmore efficient isolation of the nascent protein. These antibodiesagainst BODIPY-FL can be used for quantitation of incorporation of theBODIPY into the nascent protein.

A marker can also be modified after the tRNA molecule is aminoacylatedor misaminoacylated using chemical reactions which specifically modifythe marker without significantly altering the functional activity of theaminoacylated tRNA. These types of post-aminoacylation modifications mayfacilitate detection, isolation or purification, and can sometimes beused where the modification allow the nascent protein to attain a nativeor more functional configuration.

Fluorescent and other markers have detectable electromagnetic spectralproperties that can be detected by spectrometers and distinguished fromthe electromagnetic spectral properties of native amino acids.Spectrometers which are most useful include fluorescence, Raman,absorption, electron spin resonance, visible, infrared and ultravioletspectrometers. Other markers, such as markers with distinct electricalproperties can be detected by an apparatus such as an ammeter, voltmeteror other spectrometer. Physical properties of markers which relate tothe distinctive interaction of the marker with an electromagnetic fieldis readily detectable using instruments such as fluorescence, Raman,absorption, electron spin resonance spectrometers. Markers may alsoundergo a chemical, biochemical, electrochemical or photochemicalreaction such as a color change in response to external forces or agentssuch as an electromagnetic field or reactant molecules which allows itsdetection.

One class of fluorescent markers contemplated by the present inventionis the class of small peptides that can specifically bind to moleculeswhich, upon binding, are detectable. One example of this approach is thepeptide having the sequence of WEAAAREACCRECCARA (SEQ ID NO: 4). Thissequence (which contains four cysteine residues) allows the peptide tospecifically bind the non-fluorescent dye molecule4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FLASH, which stands forfluorescein arsenic helix binder). This dye has the interesting propertythat, upon binding, it becomes fluorescent. In other words, fluorescenceis observed only when this specific peptide sequence is present in thenascent protein. So by putting the peptide sequence at the N- orC-terminal, one can easily monitor the amount of protein synthesized.This peptide sequence can be introduced by designing the nucleic acidprimers such that they carry a region encoding the peptide sequence.

Regardless of which class of fluorescent compounds is used, detectionnormally first involves physical separation of the nascent proteins fromother biomolecules present in the cellular or cell-free proteinsynthesis system. Protein separation can be performed using, forexample, gel electrophoresis or column chromatography and can be furtherfacilitated with affinity markers which uniquely bind acceptor groups.Detection of a marker containing a fluorophore by gel electrophoresiscan be accomplished using conventional fluorescence detection methods.

After protein synthesis in a cell-free system, the reaction mixture,which contains all of the biomolecules necessary for protein synthesisas well as nascent proteins, is loaded onto a gel which may be composedof polyacrylamide or agarose (R. C. Allen et al., Gel Electrophoresisand Isoelectric Focusing of Proteins, Walter de Gruyter, New York 1984).This mixture also contains the misaminoacylated tRNAs bearing the markeras well as uncharged tRNAs. Subsequent to loading the reaction mixture,a voltage is applied which spatially separates the proteins on the gelin the direction of the applied electric field. The proteins separateand appear as a set of discrete or overlapping bands which can bevisualized using a pre- or post-gel staining technique such as Coomassieblue staining. The migration of the protein band on the gel is afunction of the molecular weight of the protein with increasing distancefrom the loading position being a function of decreasing molecularweight. Bands on the gel which contain nascent proteins will exhibitfluorescence when excited at a suitable wavelength. These bands can bedetected visually, photographically or spectroscopically and, ifdesired, the nascent proteins purified from gel sections.

For example, if BODIPY-FL is used as a marker, nascent proteins willfluoresce at 510 nm when excited by UV illumination. This fluorescencecan be detected visually by simply using a standard hand-held UVilluminator or a transilluminator. Approximately 10 nanograms (ng) ofthe protein alpha-hemolysin is detectable using this method. Also usefulare electronic imaging devices which can rapidly screen and identifyvery low concentrations of markers such as a fluorescent scanner basedon a low-temperature CCD imager. In this case as low as 0.3 ng ofprotein can be detected.

The molecular weight and quantity of the nascent protein can bedetermined by comparison of its band-position on the gel with a set ofbands of proteins of predetermined molecular weight which arefluorescently labeled. For example, a nascent protein of molecularweight 25,000 could be determined because of its relative position onthe gel relative to a calibration gel containing the commerciallyavailable standard marker proteins of known quantities and with knownmolecular weights (bovine serum albumin, 66 kD; porcine heart fumarase,48.5 kD; carbonic anhydrase, 29 kD, β-lactoglobulin, 18.4 kD;α-lactoglobulin, 14.2 kD; Sigma Chemical; St. Louis, Mo.).

Calibration proteins may also contain a similar markers for convenientdetection using the same method as the gel bearing the nascent protein.This can be accomplished in many cases by directly reacting thecalibration proteins with a molecule similar to the marker. For example,the calibration proteins can be modified with dansyl chloride so as toobtain their fluorescent derivatives (R. E. Stephens, Anal. Biochem. 65,369-79, 1975). Alternatively, the proteins could be labeled with an NHSderivitive of BODIPY-FL. These fluorescent proteins can be analyzedusing PAGE. Combined detection of these fluorescent calibration proteinsalong with that of nascent protein which contains fluorescent markeranalog will accurately determine both the molecular weight and quantityof the nascent protein synthesized. If necessary, the amounts of markerwithin each calibration and nascent protein can be determined to providean accurate quantitation. Proteins with predetermined levels offluorescent markers can be used advantageously to provide forquantitation of the gel bearing the nascent protein. This could beaccomplished by genetically engineering a calibration protein so that itcontains a specific reactive residue such as cysteine so that only onefluorescent dye Will be attached per protein.

Other methods of protein separation are also useful for detection andsubsequent isolation and purification of nascent proteins containingmarkers. For example, proteins can be separated using capillaryelectrophoresis, isoelectric focusing, low pressure chromatography andhigh-performance or fast-pressure liquid chromatography (HPLC or FPLC).In these cases, the individual proteins are separated into fractionswhich can be individually analyzed by fluorescent detectors at theemission wavelengths of the markers. Alternatively, on-line fluorescencedetection can be used to detect nascent proteins as they emerge from thecolumn fractionation system. A graph of fluorescence as a function ofretention time provides information on both the quantity and purity ofnascent proteins produced.

Another embodiment of the invention is directed to a method forlabeling, detecting and, if desired, isolating and purifying nascentproteins, as described above, containing cleavable markers. Cleavablemarkers comprise a chemical structure which is sensitive to externaleffects such as physical or enzymatic treatments, chemical or thermaltreatments, electromagnetic radiation such as gamma rays, x-rays,ultraviolet light, visible light, infrared light, microwaves, radiowaves or electric fields. The marker is aminoacylated to tRNA moleculesas before using conventional technology or misaminoacylated and added toa translation system. After incubation and production of nascentproteins, marker can be cleaved by the application of specifiedtreatments and nascent proteins detected. Alternatively, nascentproteins may also be detected and isolated by the presence or absence ofthe cleaved marker or the chemical moiety removed from the marker.

One example of a cleavable marker is a photocleavable marker such aschemical compounds which contain the 2-nitrobenzyl moiety (FIG. 6A).Upon illumination, these aromatic nitro compounds undergo an internaloxidation-reduction reaction (Pillai, Synthesis 1-26, 1980; Patchorniket al., J. Am. Chem. Soc. 92:6333-35, 1970). As a result, the nitrogroup is reduced to a nitroso group and an oxygen is inserted into thebenzylic carbon-hydrogen bond at the ortho position. The primaryphotochemical process is the intramolecular hydrogen abstraction by theexcited nitro group. This is followed by an electron-redistributionprocess to the aci-nitro form which rearranges to the nitroso product.Subsequent thermal reaction leads to the cleavage of substrate from thenitrobenzyl linkage (FIG. 6B). Examples of other cleavable markers areshown in FIG. 7.

It may sometimes be desirable to create a distance between the substrateand the cleavable moiety. To accomplish this, cleavable moieties may beseparated from substrates by cross-linker arms. Cross-linkers increasesubstrate access and stabilize the chemical structure, and can beconstructed using, for example, long alkyl chains or multiple repeatunits of caproyl moieties linked via amide linkages.

There are as many methods to synthesize cleavable markers as there aredifferent markers. One example for the synthesis of photocleavablebiotins based on nitrobenzyl alcohols involves four major steps.2-bromo-2′-nitroacetphenone, a precursor of the photoreactive moiety, isfirst converted into an α- or ω-amino acid like ε-aminocaprylic acid.The resulting acid and amino functionality of the photoreactive group iscoupled using dicyclohexyl carbodiimide (DCC). The benzoyl carbonylgroup of the resulting amide is reduced using sodium borohydride. Theresulting derivative of nitrobenzyl alcohol is derivatized to obtain thefinal component which is able to react with biomolecular substrates, forexample by the reaction with phosgene gas, to introduce thechloroformate functionality. The resulting compound is depicted in FIG.8A along with alternative derivatives of PCB. Possible linkages to aminoacids are depicted in FIG. 8B.

Cleavable markers can facilitate the isolation of nascent proteins. Forexample, one type of a cleavable marker is photocleavable biotin coupledto an amino acid. This marker can be incorporated into nascent proteinsand the proteins purified by the specific interaction of biotin withavidin or streptavidin. Upon isolation and subsequent purification, thebiotin is removed by application of electromagnetic radiation andnascent proteins utilized in useful applications without thecomplications of an attached biotin molecule. Other examples ofcleavable markers include photocleavable coumarin, photocleavabledansyl, photocleavable dinitrophenyl and photocleavable coumarin-biotin.Photocleavable markers are cleaved by electromagnetic radiation such asUV light, peptidyl markers are cleaved by enzymatic treatments, andpyrenyl fluorophores linked by disulfide bonds are cleaved by exposureto certain chemical treatments such as thiol reagents.

Cleavage of photocleavable markers is dependent on the structure of thephotoreactive moiety and the wavelength of electromagnetic radiationused for illumination. Other wavelengths of electromagnetic radiationshould not damage the proteins or other chemical moieties. In the caseof unsubstituted 2-nitrobenzyl derivatives, the yield of photolysis andrecovery of the substrate are significantly decreased by the formationof side products which act as internal light filters and are capable toreact with amino groups of the substrate. Typical illumination timesvary from 1 to about 24 hours and yields are 1-95%. Radiation sourcesare placed within about 10 cm of the substrate proteins and set on lowpower so as to minimize side reactions, if any, which may occur in thenascent proteins. In the case of alpha-substituted 2-nitrobenzylderivatives (methyl, phenyl, etc.), a considerable increase in rate ofphoto-removal as well as yield of the released substrate are observed.The introduction of other electron donor groups into phenyl rings ofphotoreactive moieties increases the yield of substrate. The generalreaction for the photolysis of PCB is depicted in FIG. 9.

For enzymatic cleavage, markers introduced contain specific bonds whichare sensitive to unique enzymes of chemical substances. Introduction ofthe enzyme or chemical into the protein mixture cleaves the marker fromthe nascent protein. When the marker is a modified amino acid, this canresult in the production of native protein forms. Thermal treatments of,for example, heat sensitive chemical moieties operate in the samefashion. Mild application of thermal energy, such as with microwaves orradiant heat, cleaves the sensitive marker from the protein withoutproducing any significant damage to the nascent proteins.

DESCRIPTION OF PREFERRED EMBODIMENTS

Nonsense or frameshift mutations, which result in a truncated geneproduct, are prevalent in a variety of disease-related genes. Den Dunnenet al., The Protein Truncation Test: A Review. Hum Mutat 14:95-102(1999). Specifically, these diseases include: i) APC (colorectalcancer), Powell et al., Molecular Diagnosis Of Familial AdenomatousPolyposis. N Engl J Med 329:1982-1987 (1993); van der Luijt et al.,Rapid Detection Of Translation-Terminating Mutations At The AdenomatousPolyposis Coli (APC) Gene By Direct Protein Truncation Test. Genomics20:1-4 (1994); Traverso et al., Detection Of APC Mutations In Fecal DNAFrom Patients With Colorectal Tumors. N Engl J Med 346:311-320 (2002);Kinzler et al., Identification Of A Gene Located At Chromosome 5q21 ThatIs Mutated In Colorectal Cancers. Science 251:1366-1370 (1991); andGroden et al., Identification And Characterization Of The FamilialAdenomatous Polyposis Coli Gene. Cell 66:589-600 (1991); ii) BRCA1 andBRCA2 (breast and ovarian cancer), Hogervosrt et al., Rapid Detection OfBRCA1 Mutations By The Protein Truncation Test. Nat Genet 10:208-212(1995); Garvin et al., A Complete Protein Truncation Test For BRAC1 andBRAC2. Eur J Hum Genet 6:226-234 (1998); Futreal et al., BRAC1 MutationsIn Primary Breast And Ovarian Carcinomas. Science 266:120-122 (1994);iii) polycystic kidney disease, Peral et al., Identification OfMutations In the Duplicated Region Of The Polycystic Kidney Disease 1Gene (PKD1) By A Novel Approach. Am J. Hum Genet 60:1399-1410 (1997);iv) neurofibromatosis (NF1 and NF2), Hein et al., Distribution Of 13Truncating Mutations In The Neurofibromatosis 1 Gene. Hum Mol Genet4:975-981 (1995); Parry et al, Germ-line Mutations In TheNeurofibromatosis 2 Gene: Correlations With Disease Severity And RetinalAbnormalities. Am J Hum Genet 59:529-539 (1996).; and v) Duchennemuscular dystrophy (DMD), Roest et al., Protein Truncation Test (PTT) ToRapidly Screen The DMD gene For Translation Terminating Mutations.Neuromuscul Disord 3:391-394 (1993). Such chain truncating mutations canbe detected using the protein truncation test (PTT). This test is basedon cell-free coupled transcription-translation of PCR (RT-PCR) amplifiedportions of the target gene (target mRNA) followed by analysis of thetranslated product(s) for shortened polypeptide fragments. However,conventional PTT is not easily adaptable to high throughput applicationssince it involves SDS-PAGE followed by autoradiography or Western blot.It is also subject to human error since it relies on visual inspectionto detect mobility shifted bands. To overcome these limitations, we havedeveloped the first high throughput solid-phase protein truncation test(HTS-PTT). HTS-PTT uses a combination of misaminoacylated tRNAs(Rothschild et al., tRNA-Mediated Protein Engineering. Curr OpinBiotechnol 10:64-70 (1999); and Gite et al, UltrasensitiveFluorescence-Based Detection Of Nascent Proteins In Gels. Anal Biochem279:218-225 (2000)), which incorporate affinity tags for surface captureof the cell-free expressed protein fragments, and specially designed PCRprimers, which introduce N- and C-terminal markers for measuring therelative level of shortened polypeptide produced by the chain truncationmutation. After cell-free translation of the protein fragments, captureand detection is accomplished in a single-well using a standard 96-wellmicrotiter plate ELISA format and chemiluminescence readout. Thetechnique is demonstrated for the detection of chain truncationmutations in the APC gene using DNA or RNA from cancer cell lines aswell as DNA of individuals pre-diagnosed with familial adenomatouspolyposis (FAP). HTP-PTT can also provide a high throughput method fornon-invasive colorectal cancer screening when used in conjunction withmethods of enriching/amplifying low-abundance mutant DNA. Traverso etal. (2002).

A. Detection of Mutations

Detection of mutations is an increasingly important area in clinicaldiagnosis, including but not limited to the diagnosis of cancer and/orindividuals disposed to cancer. The protein truncation test (PTT) is atechnique for the detection of nonsense and frameshift mutations whichlead to the generation of truncated protein products. Genes associatedwith Duchenne muscular dystrophy, adenomatous polyposis coli, human mutLhomologue and human nutS homologue (both involved in colon cancer), andBRAC1 (involved in familial breast cancer) can now be screened formutations in this manner, along with others (see Table 1).

Typically, the PTT technique involves the incorporation of a T7 promotersite, ribosome binding site, and an artificial methionine start siteinto a PCR product covering the region of the gene to be investigated.The PCR product is then transcribed and translated using either an invitro rabbit reticulocyte lysate or wheat germ lysate system, togenerate a protein corresponding to the region of the gene amplified.The presence of a stop codon in the sequence, generated by a nonsensemutation or a frameshift, will result in the premature termination ofprotein translation, producing a truncated protein that can be detectedby standard gel electrophoresis (e.g. SDS-PAGE) analysis combined withradioactive detection.

There are drawbacks to the technique as currently practiced. One of themost important problems involves the identification of the product ofinterest. This is made difficult because of nonspecific radiolabeledproducts. Attempts to address these problems have been made. Oneapproach is to introduce an affinity tag after the start site and beforethe region encoding the gene of interest. See Rowan and Bodmer,“Introduction of a myc Reporter Taq to Improve the Quality of MutationDetection Using the Protein Truncation Test,” Human Mutation 9:172(1997). However, such approaches still have the disadvantage that theyrely on electrophoresis.

The present invention contemplates a gel-free truncation test (GFTT),wherein two or three markers are introduced into the nascent protein.The present invention contemplates both pre-natal and post-natal testingto determine predisposition to disease. In a preferred embodiment of theinvention, the novel compositions and methods are directed to thedetection of frameshift or chain terminating mutations. In order todetect such mutations, a nascent protein is first synthesized in acell-free or cellular translation system from message RNA or DNA codingfor the protein which may contain a possible mutation. The nascentprotein is then separated from the cell-free or cellular translationsystem using an affinity marker located at or close to the

TABLE 1 Applications of PTT in Human Molecular Genetics DiseaseReferences % Truncating Mutations Gene Familial Adenomatous 95% APCPolyposis Hereditary desmold disease 100% APC Ataxia telangiectasia 90%ATM Hereditary Breast and 90% BRCA1 Ovarian Cancer 90% BRCA2 CysticFibrosis 15% CFTR Duchenne Muscular 95% DMD Dystrophy Emery-DreifussMuscular 80% EMD Dystrophy Fanconi anaemia 80% FAA Hunter Syndrome −50%IDS Hereditary non-polyposis −80% hMSH2 colorectal cancer −70% hMLH1Neurofibromatosis type 1 50% NF1 Neurofibromatosis type 2 65% NF2Polycystic Kidney Disease 95% PKD1 Rubinstein-Taybi Syndrome 10% RTS Thepercentage of truncating mutations reported which should be detectableusing PTT.N-terminal end of the protein. The protein is then analyzed for thepresence of a detectable marker located at or close to the N-terminal ofthe protein (N-terminal marker). A separate measurement is then made ona sequence dependent detectable marker located at or close to theC-terminal end of the protein (C-terminal marker).

A comparison of the measurements from the C-terminal marker andN-terminal marker provides information about the fraction of nascentproteins containing frameshift or chain terminating mutations in thegene sequence coding for the nascent protein. The level of sequencedependent marker located near the C-terminal end reflects the fractionof protein which did not contain chain terminating or out-of-framemutations. The measurement of the N-terminal marker provides an internalcontrol to which measurement of the C-terminal marker is normalized.Normalizing the level of the C-terminal marker to the N-terminal markereliminates the inherent variabilities such as changes in the level ofprotein expression during translation that can undermine experimentalaccuracy. Separating the protein from the translation mixture using anusing an affinity marker located at or close to the N-terminal end ofthe protein eliminates the occurrence of false starts which can occurwhen the protein is initiated during translation from an internal AUG inthe coding region of the message. A false start can lead to erroneousresults since it can occurs after the chain terminating or out-of-framemutation. This is especially true if the internal AUG is in-frame withthe message. In this case, the peptide C-terminal marker will still bepresent even if message contains a mutation.

In one example, a detectable marker comprising a non-native amino acidor amino acid derivative is incorporated into the nascent protein duringits translation at the amino terminal (N-terminal end) using amisaminoacylate initiator tRNA which only recognizes the AUG start codonsignaling the initiation of protein synthesis. One example of adetectable marker is the highly fluorescent compound BODIPY FL. Themarker might also be photocleavable such as photocleavable coumarin orphotocleavable biotin. The nascent protein is then separated from thecell-free or cellular translation system by using a coupling agent whichbinds to an affinity marker located adjacent to the N-terminal of theprotein. One such affinity marker is a specific protein sequence knownas an epitope. An epitope has the property that it selectively interactswith molecules and/or materials containing acceptor groups. There aremany epitope sequences reported in the literature including H is ×6(HHHHHH) (SEQ ID NO: 5) described by ClonTech and C-myc (−EQKLISEEDL)(SEQ ID NO:6) described by Roche-BM, Flag (DYKDDDDK) (SEQ ID NO:7)described by Stratagene), SteptTag (WSHPQFEK) (SEQ ID NO:8) described bySigma-Genosys and HA Tag (YPYDVPDYA) (SEQ ID NO:9) described byRoche-BM.

Once the nascent protein is isolated from the translation system, it isanalyzed for presence of the detectable marker incorporated at theN-terminal of the protein. In the case of BODIPY FL, it can be detectedby measuring the level of fluorescence using a variety of commerciallyavailable instrument such as a Molecular Dynamics Model 595 fluorescencescanner that is equipped with excitation at 488 nm and an emissionfilter that allows light above 520 nm to be transmitted to the detector.

The protein is then analyzed for the presence of a sequence specificmarker located near the C-terminal end of the protein. In normalpractice, such a sequence specific marker will consist of a specificsequence of amino acids located near the C-terminal end of the proteinwhich is recognized by a coupling agent. For example, an antibody can beutilized which, is directed against an amino acid sequence located at ornear C-terminal end of the nascent protein can be utilized. Suchantibodies can be labeled with a variety of markers includingfluorescent dyes that can be easily detected and enzymes which catalyzedetectable reactions that lead to easily detectable substrates. Themarker chosen should have a different detectable property than that usedfor the N-terminal marker. An amino acid sequence can also comprise anepitope which is recognized by coupling agents other than antibodies.One such sequence is 6 histidines sometimes referred to as a his-tagwhich binds to cobalt complex coupling agent.

A variety of N-terminal markers, affinity markers and C-terminal markersare available which can be used for this embodiment. The N-terminalmarker could be BODIPY, affinity marker could be StrepTag and C-terminalmarker could be a His×6 tag. In this case, after translation, thereaction mixture is incubated in streptavidin coated microtiter plate orwith streptavidin coated beads. After washing unbound material, theN-terminal marker is directly measured using a fluorescence scannerwhile the C-terminal marker can be quantitated using anti-his×6antibodies conjugated with a fluorescent dye (like rhodamine or TexasRed) which has optical properties different than BODIPY, thusfacilitating simultaneous dual detection.

In a different example, the N-terminal marker could be a biotin orphotocleavable biotin incorporated by a misaminoacylated tRNA, theaffinity marker could be a His ×6 tag and the C-terminal had C-mycmarker. In this case, after the translation, the reaction mixture isincubated with metal chelating beads or microtiter plates (for exampleTalon, ClonTech). After washing the unbound proteins, the plates orbeads can be subjected to detection reaction using streptavidinconjugated fluorescence dye and C-myc antibody conjugated with otherfluorescent dye. In addition, one can also use chemiluminescentdetection method using antibodies which are conjugated with peroxidases.

It will be understood by those skilled in the area of molecular biologyand biochemistry that the N-terminal marker, affinity marker andC-terminal marker can all consist of epitopes that can be incorporatedinto the nascent protein by designing the message or DNA coding for thenascent protein to have a nucleic acid sequence corresponding to theparticular epitope. This can be accomplished using known methods such asthe design of primers that incorporate the desired nucleic acid sequenceinto the DNA coding for the nascent protein using the polymerase chainreaction (PCR). It will be understood by those skilled in proteinbiochemistry that a wide variety of detection methods are available thatcan be used to detect both the N-terminal marker and the C-terminalmarkers. Additional examples include the use of chemiluminescence assayswhere an enzyme which converts a non-chemiluminescent substrate to achemiluminescent product is conjugated to an antibody that is directedagainst a particular epitope.

One example of this approach is based on using a luminometer to measureluminescent markers. A biotin detectable marker is incorporated at theN-terminal using a misaminoacylated tRNA. The biotin is detected byusing a streptavidin which is conjugated to Renilla luciferase from seapansy. The C-terminal sequence comprises an epitope which interacts witha binding agent that has attached firefly luciferase. After separationof the nascent protein using a distinct epitope located near theN-terminal end of the protein, the protein is subjected to a dualluminescent luciferase assay based on a procedure described by PromegaCorp and known as the Dual-Luciferase® Reporter Assay. This assayconsists of first adding Luciferase Assay Reagent II available fromPromega Corp. to the isolated nascent protein and then measuring thelevel of chemiluminescence. Stop & Glo® Reagent is then added whichsimultaneously quenches the firefly luminescence and activates theRenilla luminescence. The luciferase assay can be performed andquantified in seconds. A comparison of the level of luminescencemeasured from the firefly and Renilla luciferase provides an indicationof whether a mutation is present or not in the coding message of thenascent protein.

In an additional example, the N-terminal marker comprises an affinitymarker which is incorporated at the N-terminal end of the protein duringits translation using a misaminoacylated tRNA. The affinity markerinteracts with a coupling agent which acts to separate the nascentprotein from the translation mixture. The nascent protein also containsa detectable marker which is located adjacent or close to the N-terminalof the protein containing the affinity marker. In addition, it containsa sequence specific marker at or near the C-terminal end of the protein.The detectable markers near the N-terminal and C-terminal ends of thenascent protein are then measured and compared to detect the presence ofchain terminating or out-of-frame mutations.

There are a variety of additional affinity markers, N-terminal markersand C-terminal markers available for this embodiment. The affinitymarker could be biotin or photocleavable biotin, N-terminal marker couldbe StepTag and C-terminal the C-myc epitope. In this case, after thetranslation, the reaction mixture is incubated with streptavidin coatedbeads or microtiter plates coated with streptavidin. After washing theunbound proteins, the plates or beads can be subjected to detectionreaction using anti-his 6 antibodies conjugated with a fluorescent dye(like rhodamine or Texas Red) and C-myc antibody conjugated with otheranother fluorescent dye such as BODIPY. In addition, one can also usechemiluminescent detection method using antibodies which are conjugatedwith peroxidases. Even in case of peroxidases conjugated antibodies, onecan use fluorescent substrates and use FluorImager like device toquantitate N-terminal and C-terminal labels.

For optimal effectiveness, the N-terminal marker and affinity markershould be placed as close as possible to the N-terminal end of theprotein. For example, if an N-terminal marker is incorporated using amisaminoacylated initiator, it will be located at the N-terminal aminoacid. In this case, the affinity marker should be located immediatelyadjacent to the N-terminal marker. Thus, if a BODIPY marker whichconsists of a BODIPY conjugated to methionine is incorporated by amisaminoacylated initiator tRNA, it should be followed by an epitopesequence such as SteptTag (WSHPQFEK) (SEQ ID NO:8) so that the entireN-terminal sequence will be BODIPY-MWSPQFEK (SEQ ID NO: 10). However,for specific cases it may be advantageous to add intervening amino acidsbetween the BODIPY-M and the epitope sequence in order to avoidinteraction between the N-terminal marker and the affinity marker or thecoupling agent which binds the affinity marker. Such interactions willvary depending on the nature of the N-terminal marker, affinity markerand coupling agent.

For optimal effectiveness, the C-terminal marker should be placed asclose as possible to the C-terminal end of protein. For example, if aHis-X6 tag is utilized, the protein sequence would terminate with 6 His.In some cases, an epitope may be located several residues before theC-terminal end of the protein in order to optimize the properties of thenascent protein. This might occur for example, if a specific amino acidsequence is necessary in order to modify specific properties of thenascent protein that are desirable such as its solubility orhydrophobicity.

In the normal application of this method, the ratio of the measuredlevel of N-terminal and C-terminal markers for a nascent proteintranslated from a normal message can be used to calculate a standardnormalized ratio. In the case of a message which may contain a mutation,deviations from this standard ratio can then be used to predict theextent of mutations. For example, where all messages are defective, theratio of the C-terminal marker to the N-terminal marker is expected tobe zero. On the other hand, in the case where all messages are normal,the ratio is expected to be 1. In the case where only half of themessage is defective, for example for a patient which is heterozygotefor a particular genetic defect which is chain terminating or causes anout-of-frame reading error, the ratio would be ½.

There are several unique advantages of this method compared to existingtechniques for detecting chain terminating or out-of-frame mutations.Normally, such mutations are detected by analyzing the entire sequenceof the suspect gene using conventional DNA sequencing methods. However,such methods are time consuming, expensive and not suitable for rapidthroughput assays of large number of samples. An alternative method isto utilize gel electrophoresis, which is able to detect changes from theexpected size of a nascent protein. This approach, sometimes referred toas the protein truncation test, can be facilitated by usingnon-radioactive labeling methods such as the incorporation of detectablemarkers with misaminoacylated tRNAs. However, in many situations, suchas high throughput screening, it would be desirable to avoid the use ofgel electrophoresis which is time-consuming (typically 60-90 minutes).In the present method, the need for performing gel electrophoresis iseliminated. Furthermore, since the approach depends on comparison of twodetectable signals from the isolated nascent protein which can befluorescent, luminescent or some combination thereof, it is highlyamenable to automation.

Measuring a sequence dependent marker located near the C-terminal end ofthe protein provides information about the presence of either aframeshift or chain terminating mutation since the presence of eitherwould result in an incorrect sequence. The measurement of the N-terminalmarker provides an internal control to which measurement of theC-terminal marker is normalized. Normalizing the level of the C-terminalmarker to the N-terminal marker eliminates the inherent variabilitiessuch as changes in the level of protein expression during translationthat can undermine experimental accuracy. Separating the protein fromthe translation mixture using an using an affinity marker located at orclose to the N-terminal end of the protein eliminates the occurrence offalse starts which can occur when the protein is initiated duringtranslation from an internal AUG in the coding region of the message. Afalse start can lead to erroneous results since it can occurs after thechain terminating or out-of-frame mutation. This is especially true ifthe internal AUG is in-frame with the message. In this case, the peptideC-terminal marker will still be present even if message contains amutation.

B. Reporter Groups

Another embodiment of the invention is directed to a method formonitoring the synthesis of nascent proteins in a cellular or acell-free protein synthesis system without separating the components ofthe system. These markers have the property that once incorporated intothe nascent protein they are distinguishable from markers free insolution or linked to a tRNA. This type of marker, also called areporter, provides a means to detect and quantitate the synthesis ofnascent proteins directly in the cellular or cell-free translationsystem.

One type of reporters previously described in U.S. Pat. No. 5,643,722(hereby incorporated by reference) has the characteristic that onceincorporated into the nascent protein by the protein synthesizingsystem, they undergo a change in at least one of their physical orphysio-chemical properties. The resulting nascent protein can beuniquely detected inside the synthesis system in real time without theneed to separate or partially purify the protein synthesis system intoits component parts. This type of marker provides a convenientnon-radioactive method to monitor the production of nascent proteinswithout the necessity of first separating them from pre-existingproteins in the protein synthesis system. A reporter marker would alsoprovide a means to detect and distinguish between different nascentproteins produced at different times during protein synthesis byaddition of markers whose properties are distinguishable from eachother, at different times during protein expression. This would providea means of studying differential gene expression.

One example of the utilization of reporters is schematically representedin FIG. 10. A tRNA molecule is misaminoacylated with a reporter (R)which has lower or no fluorescence at a particular wavelength formonitoring and excitation. The misaminoacylated tRNA is then introducedinto a cellular or cell-free protein synthesis system and the nascentproteins containing the reporter analog are gradually produced. Uponincorporation of the reporter into the nascent protein (R*), it exhibitsan increased fluorescence at known wavelengths. The gradual productionof the nascent protein is monitored by detecting the increase offluorescence at that specific wavelength.

The chemical synthesis of a reporter can be based on the linkage of achemical moiety or a molecular component having reporter properties witha native amino acid residue. There are many fluorescent molecules whichare sensitive to their environment and undergo a change in thewavelength of emitted light and yield of fluorescence. When thesechemical moieties, coupled to amino acids, are incorporated into thesynthesized protein, their environments are altered because of adifference between the bulk aqueous medium and the interior of a proteinwhich can causes reduced accessibility to water, exposure to chargedionic groups, reduced mobility, and altered dielectric constant of thesurrounding medium. Two such examples are shown in FIG. 11.

One example of a reporter molecule is based on a fluorescent acridiniummoiety and has the unique property of altering its emission properties,depending upon polarity or viscosity of the microenvironment. It has ahigher quantum yield of fluorescence when subjected to hydrophobicenvironment and/or viscosity. Due to the hydrophobicity of the reporteritself, it is more likely to be associated with the hydrophobic core ofthe nascent protein after incorporation into the growing nascentpolypeptide. An increase in the fluorescence intensity is a directmeasure of protein synthesis activity of the translation system.Although, the environment of each reporter residue in the protein willbe different, and in some cases, the reporter may be present on thesurface of the protein and exposed to an aqueous medium, a net changeoccurs in the overall spectroscopic properties of the reportersincorporated into the protein relative to bulk aqueous medium. A changein the spectroscopic properties of only a subset of reporters in theprotein will be sufficient to detect the synthesis of proteins thatincorporate such reporters.

An alternative approach is to utilize a reporter which alters itsfluorescent properties upon formation of a peptide bond and notnecessarily in response to changes in its environment. Changes in thereporter's fluorescence as it partitions between different environmentsin the cell-free extract does not produce a large signal change comparedto changes in fluorescence upon incorporation of the reporter into thenascent protein.

A second example of a reporter is a marker based on coumarmn such as6,7-(4′,5′-prolino)coumarin. This compound can be chemically synthesizedby coupling a fluorophore like coumarin with an amino-acid structuralelement in such a way that the fluorophore would alter its emission orabsorption properties after forming a peptide linkage (FIG. 11). Forexample, a proline ring containing secondary amino functions willparticipate in peptide bond formation similar to a normal primary aminogroup. Changes in fluorescence occur due to the co-planarity of thenewly formed peptide group in relation to the existing fluorophore. Thisincreases conjugation/delocalization due to the π-electrons ofnitrogen-lone pair and carbonyl-group in the peptide bond. Synthesis ofsuch compounds is based on coumarin synthesis using ethylacetoacetate(FIG. 11).

Reporters are not limited to those non-native amino acids which changetheir fluorescence properties when incorporated into a protein. Thesecan also be synthesized from molecules that undergo a change in otherelectromagnetic or spectroscopic properties including changes inspecific absorption bands in the UV, visible and infrared regions of theelectromagnetic spectrum, chromophores which are Raman active and can beenhanced by resonance Raman spectroscopy, electron spin resonanceactivity and nuclear magnetic resonances. In general, a reporter can beformed from molecular components which undergo a change in theirinteraction and response to electromagnetic fields and radiation afterincorporation into the nascent protein.

In the present invention, reporters may also undergo a change in atleast one of their physical or physio-chemical properties due to theirinteraction with other markers or agents which are incorporated into thesame nascent protein or are present in the reaction chamber in which theprotein is expressed. The interaction of two different markers with eachother causes them to become specifically detectable. One type ofinteraction would be a resonant energy transfer which occurs when twomarkers are within a distance of between about 1 angstrom (A) to about50 A, and preferably less than about 10 A. In this case, excitation ofone marker with electromagnetic radiation causes the second marker toemit electromagnetic radiation of a different wavelength which isdetectable. A second type of interaction would be based on electrontransfer between the two different markers which can only occur when themarkers are less than about 5 A. A third interaction would be aphotochemical reaction between two markers which produces a new speciesthat has detectable properties such as fluorescence. Although thesemarkers may also be present on the misaminoacylated tRNAs used for theirincorporation into nascent proteins, the interaction of the markersoccurs primarily when they are incorporated into protein due to theirclose proximity. In certain cases, the proximity of two markers in theprotein can also be enhanced by choosing tRNA species that will insertmarkers into positions that are close to each other in either theprimary, secondary or tertiary structure of the protein. For example, atyrosine-tRNA and a tryptophan-tRNA could be used to enhance theprobability for two different markers to be near each other in a proteinsequence which contains the unique neighboring pair tyrosine-tryptophan.

In one embodiment of this method, a reporter group is incorporated intoa nascent protein using a misaminoacylated tRNA so that when it binds toa coupling agent, the reporter group interacts with a second markers oragents which causes them to become specifically detectable. Such aninteraction can be optimized by incorporating a specific affinityelement into the nascent protein so that once it interacts with acoupling agent the interaction between the reporter group and the secondmarker is optimized. Such an affinity element might comprise a specificamino acid sequence which forms an epitope or a normative amino acid. Inone example, the reporter group is incorporated at the N-terminal of thenascent protein by using a misaminoacylated tRNA. The epitope isincorporated into the nascent protein so that when it interacts with thecoupling agent the reporter comes into close proximity with a secondmarker which is conjugated to the coupling agent.

One type of interaction between the markers that is advantageously usedcauses a fluorescence resonant energy transfer which occurs when the twomarkers are within a distance of between about 1 angstrom (A) to about50 A, and preferably less than about 10 A. In this case, excitation ofone marker with electromagnetic radiation causes the second marker toemit electromagnetic radiation of a different wavelength which isdetectable. This could be accomplished, for example, by incorporating afluorescent marker at the N-terminal end of the protein using the E.coli initiator tRNA^(fmet). An epitope is then incorporated near theN-terminal end such as the SteptTag (WSHPQFEK) (SEQ ID NO:8) describedby Sigma-Genosys. Streptavidin is then conjugated using known methodswith a second fluorescent marker which is chosen to efficiently undergofluorescent energy transfer with marker 1. The efficiency of thisprocess can be determined by calculating the a Forster energy transferradius which depends on the spectral properties of the two markers. Themarker-streptavidin complex is then introduced into the translationmixture. Only when nascent protein is produced does fluorescent energytransfer between the first and second marker occur due to the specificinteraction of the nascent protein StreptTag epitope with thestreptavidin.

In one preferred embodiment of the invention a fluorescent reportergroup is incorporated into a nascent protein which is partially or fullyquenched when misaminoacylating a tRNA. This is achieved by engineeringthe tRNA so that it contains a second group which effectively quenchesthe fluorescent reporter group (i.e. the quencher group). In oneexample, this method contemplates an initiator tRNA because of thepresence of a modified base (4-thio-uridine; 4s-U) at position 8. (seeFIG. 66) This modified base acts as a reactive group to which thequencher molecule can be attached. The quencher modified tRNA is thenmisaminoacylated with a methionine followed by coupling of thefluorescent reporter group to the NH₂ group of methionine. Similar tophenylalanine-tRNA (Phe-tRNA), modification with a fluorophore of a 4s-Unucleotide at base position 8 should not interfere significantly withthe aminoacylation of the initiator tRNA (Johnson et al., 156:113-140(1982)). With the proper selection of quencher and reporter molecules,the misaminoacylated tRNA can be designed to have minimum fluorescenceof the reporter since the quencher and reporter are separated by adistance (e.g. 20-30 Å) in the range where quenching is effective. Incontrast, once the reporter group is transferred from the tRNA to thegrowing nascent protein, it will exhibit fluorescence due to theseparation of the quencher and reporter group (e.g. the donor andacceptor group). Furthermore, the fluorescence will be directlyproportional to the amount of nascent protein produced by the in vitroexpression system.

Preparation of FO-tRNA: The initiator tRNA^(fmet) is first modified withquencher molecule, QSY7-malemide. The typical reactions contained 1.5nanomoles (˜1.0 OD₂₆₀) of tRNA in 50 mM potassium phosphate buffer, pH8.4 and will be incubated with 150 nanomoles QSY7-malemide for 4-5 hoursat room temperature in dark. After the modification reaction, theunincorporated dye is removed by G-25 desalting column and the modifiedtRNA (Q-tRNA) is ethanol precipitated. The Q-tRNA is aminoacylated withmethionine using an excess of aminoacyl tRNA-synthetases under standardaminoacylation conditions. After aminoacylation, this tRNA is modifiedwith BODIPY-FL as follows: To the aminoacylated tRNA, 2.5 μl of 1NNaHCO₃ is added (final conc. 50 mM, pH 8.5) followed by 10 μl of 10 mMsolution of BODIPY-FL-SSE. The mixture is incubated for 10 min at 0° C.and the reaction is quenched by the addition of lysine (finalconcentration, 100 mM). To the resulting solution, 0.1 volume of 3 MNaOAc, pH 5.0 is added, and the modified tRNA is precipitated with 3volumes of ethanol. The precipitate is dissolved in 50 μl of water andpurified on a Sephadex G-25 gel filtration column (0.5×5 cm) to removeany free fluorescent reagent. The modified tRNA (FQ-tRNA) is storedfrozen (−70° C.) in small aliquots in order to avoid repeatedfreeze-thaws. The extent of modification of the aminoacylated-tRNA isdetermined by analytical HPLC.

Cell-free synthesis of proteins and their detection: For prokaryotictranslation mix, the typical translation reaction mixture (10 ul)contains 3 μl of extract, 4 μl of premix, 1 μl of complete amino acidmix, 30 picomoles of FQ-tRNA and 0.5 μg of the appropriate plasmid DNA.The translation reaction is incubated for 30 min at 37° C. Foreukaryotic translation mix, the typical translation reaction mixture (10μl) contains either 8 μl of rabbit reticulocyte or wheat germtranslation extract, 0.5 μl of complete amino acid mix, 30 picomoles ofFQ-tRNA and 0.5 μg of the appropriate plasmid DNA. The translationreaction is incubated for 30 min at 30° C. For SDS-PAGE, 4-10 μl aliquotof the reaction mix is precipitated with 5 volumes of acetone and theprecipitated proteins are collected by centrifugation. The pellet isdissolved in 1×SDS-gel loading buffer (62 mM Tris-HCl, 2% SDS, 10% v/vglycerol, 100 mM DTT, 0.01% bromophenol blue, pH 6.8) and subjected toSDS-PAGE after boiling for 5 min. When less than 2 μl of the extract isanalyzed, acetone precipitation step is not necessary. Afterelectrophoresis, polyacrylamide gels are scanned using a FluorImager 595(Molecular Dynamics, Sunnyvale, Calif.) equipped with an argon laser(488 nm line) as an excitation source and a 530±30 nm emission filter.Alternatively, the nascent proteins in polyacrylamide gels are detectedusing an UV-transilluminator combined with a Polaroid camera fitted witha green filter (Tiffen #58, Polaroid, Cambridge, Mass.). The reactioncarried out in absence of any added DNA is taken as negative control.

Real-Time Monitoring of Protein Synthesis: The translation is carriedout in the 96-well clear bottom black low-binding microtiter plate(Dynex Technologies, Chantilly, Va.) as explain above. The proteinsynthesis is initiated by the addition of the DNA and the plate will becontinuously read using a FluorImager SI (Molecular Dynamics, Sunnyvale,Calif.) equipped with an argon laser (488 nm line) as an excitationsource and a 530±30 nm emission filter at every 2 minutes for the periodof 30 min. The reaction carried out in absence of any added DNA is takenas negative control.

Potential results: In general the crystal structures of some of thetRNAs are known which allows one to choose the position forincorporating of a quencher molecules in order to obtain a highefficiency quenching (FIG. 67). There are a variety of quencher reporterpairs known that can be utilized and listed in FIG. 68 along with theirR₀.

The criteria for the selection of a reporter group (acceptor) includesmall size, high fluorescence quantum yield, photo-stability andinsensitivity to environment. The criteria for choosing a quenchermolecules are minimal background when both molecules (F and Q) arepresent on the tRNA molecules and its availability in suitable reactiveform. In one embodiment, the quencher is QSY7 as shown in FIG. 69.

There are a variety of dyes which can be used as marker pairs in thismethod that will produce easily detectable signals when brought intoclose proximity. Previously, such dye pairs have been used for exampleto detect PCR products by hybridizing to probes labeled with a dye onone probe at the 5′-end and another at the 3′-end. The production of thePCR product brings a dye pair in close proximity causing a detectableFRET signal. In one application the dyes, fluorescein and LC 640 wereutilized on two different primers (Roche MolecularBiochemicals-http://www.biochem.boehringer-mannheim.com/lightcycler/monitoO3.htm).When the fluorescein is excited by green light (around 500 nm) that isproduced by a diode laser, the LC 640 emits red fluorescent, light(around 640 nm) which can be easily detected with an appropriate filterand detector. In the case of nascent proteins, the pair of dyes BODIPYFL and LC 640 would function in a similar manner. For example,incorporation of the BODIPY FL on the N-terminal end of the protein andthe labeling of a binding agent with LC 640 which is directed against anN terminal epitope would allow detection of the production of nascentproteins.

The use of the marker pair BODIPY-FL and coumarin is a second pair whichcan be utilized advantageously. In one study, [Keller, R. C., Silvius,J. R., and De Kruijff, B. (1995) Biochem Biophys Res Commun 207(2),508-14] it was found using the spectral overlap a Forster energytransfer radius (RO) of 50+/−2 A and 40+/−2 A for thecoumarin-(beta-BODIPY FL) and the coumarin-(beta-BODIPY 530/550) couplerespectively. Experimentally this was estimated to be 49.0-51.5 A and38.5-42.5 A respectively. It is also possible to use two markers withsimilar or identical spectral properties for the marker pair due to theprocess of quenching. For example, in one study this process was used inthe case of BODIPY FL in order to study the processing of exogenousproteins in intact cells [Reis, R. C., Sorgine, M. H., andCoelho-Sampaio, T. (1998) Eur J Cell Biol 75(2), 192-7] and in a secondcase to study the kinetics of intracellular viral assembly [Da Poian, A.T., Gomes, A. M., and Coelho-Sampaio, T. (1998) J Virol Methods 70(1),45-58].

As stated above, a principal advantage of using reporters is the abilityto monitor the synthesis of proteins in cellular or a cell-freetranslation systems directly without further purification or isolationsteps. Reporter markers may also be utilized in conjunction withcleavable markers that can remove the reporter property at will. Suchtechniques are not available using radioactive amino acids which requirean isolation step to distinguish the incorporated marker from theunincorporated marker. With in vitro translation systems, this providesa means to determine the rate of synthesis of proteins and to optimizesynthesis by altering the conditions of the reaction. For example, an invitro translation system could be optimized for protein production bymonitoring the rate of production of a specific calibration protein. Italso provides a dependable and accurate method for studying generegulation in a cellular or cell-free systems.

C. Affinity Markers

Another embodiment of the invention is directed to the use of markersthat facilitate the detection or separation of nascent proteins producedin a cellular or cell-free protein synthesis system. Such markers aretermed affinity markers and have the property that they selectivelyinteract with molecules and/or materials containing acceptor groups. Theaffinity markers are linked by aminoacylation to tRNA molecules in anidentical manner as other markers of non-native amino acid analogs andderivatives and reporter-type markers as described. These affinitymarkers are incorporated into nascent proteins once the misaminoacylatedtRNAs are introduced into a translation system.

An affinity marker facilities the separation of nascent proteins becauseof its selective interaction with other molecules which may bebiological or non-biological in origin through a coupling agent. Forexample, the specific molecule to which the affinity marker interacts,referred to as the acceptor molecule, could be a small organic moleculeor chemical group such as a sulfhydryl group (—SH) or a largebiomolecule such as an antibody. The binding is normally chemical innature and may involve the formation of covalent or non-covalent bondsor interactions such as ionic or hydrogen bonding. The binding moleculeor moiety might be free in solution or itself bound to a surface, apolymer matrix, or a reside on the surface of a substrate. Theinteraction may also be triggered by an external agent such as light,temperature, pressure or the addition of a chemical or biologicalmolecule which acts as a catalyst.

The detection and/or separation of the nascent protein and otherpreexisting proteins in the reaction mixture occurs because of theinteraction, normally a type of binding, between the affinity marker andthe acceptor molecule. Although, in some cases some incorporatedaffinity marker will be buried inside the interior of the nascentprotein, the interaction between the affinity marker and the acceptormolecule will still occur as long as some affinity markers are exposedon the surface of the nascent protein. This is not normally a problembecause the affinity marker is distributed over several locations in theprotein sequence.

Affinity markers include native amino acids, non-native amino acids,amino acid derivatives or amino acid analogs in which a coupling agentis attached or incorporated. Attachment of the coupling agent to, forexample, a non-native amino acid may occur through covalentinteractions, although non-covalent interactions such as hydrophilic orhydrophobic interactions, hydrogen bonds, electrostatic interactions ora combination of these forces are also possible. Examples of usefulcoupling agents include molecules such as haptens, immunogenicmolecules, biotin and biotin derivatives, and fragments and combinationsof these molecules. Coupling agents enable the selective binding orattachment of newly formed nascent proteins to facilitate theirdetection or isolation. Coupling agents may contain antigenic sites fora specific antibody, or comprise molecules such as biotin which is knownto have strong binding to acceptor groups such as streptavidin. Forexample, biotin may be covalently linked to an amino acid which isincorporated into a protein chain. The presence of the biotin willselectively bind only nascent proteins which incorporated such markersto avidin molecules coated onto a surface. Suitable surfaces includeresins for chromatographic separation, plastics such as tissue culturesurfaces for binding plates, microtiter dishes and beads, ceramics andglasses, particles including magnetic particles, polymers and othermatrices. The treated surface is washed with, for example, phosphatebuffered saline (PBS), to remove non-nascent proteins and othertranslation reagents and the nascent proteins isolated. In some casethese materials may be part of biomolecular sensing devices such asoptical fibers, chemfets, and plasmon detectors.

One example of an affinity marker is dansyllysine (FIG. 5). Antibodieswhich interact with the dansyl ring are commercially available (SigmaChemical; St. Louis, Mo.) or can be prepared using known protocols suchas described in Antibodies: A Laboratory Manual (E. Harlow and D. Lane,editors, Cold Spring Harbor Laboratory Press, 1988) which is herebyspecifically incorporated by reference. Many conventional techniquesexist which would enable proteins containing the dansyl moiety to beseparated from other proteins on the basis of a specific antibody-dansylinteraction. For example, the antibody could be immobilized onto thepacking material of a chromatographic column. This method, known asaffinity column chromatography, accomplishes protein separation bycausing the target protein to be retained on the column due to itsinteraction with the immobilized antibody, while other proteins passthrough the column. The target protein is then released by disruptingthe antibody-antigen interaction. Specific chromatographic columnmaterials such as ion-exchange or affinity Sepharose, Sephacryl,Sephadex and other chromatography resins are commercially available(Sigma Chemical; St. Louis, Mo.; Pharmacia Biotech; Piscataway, N.J.).

Separation can also be performed through an antibody-dansyl interactionusing other biochemical separation methods such as immunoprecipitationand immobilization of the antibodies on filters or other surfaces suchas beads, plates or resins. For example, protein could be isolated bycoating magnetic beads with a protein-specific antibody. Beads areseparated from the extract using magnetic fields. A specific advantageof using dansyllysine as an affinity marker is that once a protein isseparated it can also be conveniently detected because of itsfluorescent properties.

In addition to antibodies, other biological molecules exist whichexhibit equally strong interaction with target molecules or chemicalmoieties. An example is the interaction of biotin and avidin. In thiscase, an affinity analog which contains the biotin moiety would beincorporated into the protein using the methods which are part of thepresent invention. Biotin-lysine amino acid analogs are commerciallyavailable (Molecular Probes; Eugene, Oreg.).

Affinity markers also comprise one component of a multicomponent complexwhich must be formed prior to detection of the marker. One particularembodiment of this detection means involves the use of luminescent metalchelates, in particular luminescent rare earth metal chelates. It iswell known that certain molecules form very stable complexes with rareearth metals. It is also well known that introduction of chromophoreinto these chelates sensitizes luminescence of these complexes. Avariety of detection schemes based on the use of luminescent rare earthmetal chelates have been described: Hemmila, I. A., “Applications ofFluorescence in Immunoassays”, (Wiley&Sons 1991).

In a preferred embodiment, tRNA is misaminoacylated with a chromophorethat also acts as a rare earth shelter. This modified aminoacyl tRNA isthen introduced into cellular or cell-free protein translation systemand the modified amino acid incorporated into nascent protein. Themixture is then separated using gel electrophoresis and the gel isincubated with a solution containing rare earth cation. Under theseconditions rare earth cations form luminescent complexes with aminoacids modified with a chelator present only in nascent proteins. Thenascent proteins are then detected using a mid-range UV transilluminator(350 nm), which excites the formed lanthamide complex. The image is thenrecorded using polaroid camera or CCD array and a filter. In oneembodiment, the derivatives of salicylic acid as one component andterbium ions as a second component of the binary detection system areused.

Affinity markers can also comprise cleavable markers incorporating acoupling agent. This property is important in cases where removal of thecoupled agent is required to preserve the native structure and functionof the protein and to release nascent protein from acceptor groups. Insome cases, cleavage and removal of the coupling agent results inproduction of a native amino acid. One such example is photocleavablebiotin coupled to an amino acid.

Photocleavable biotin contains a photoreactive moiety which comprises aphenyl ring derivatized with functionalities represented in FIG. 12 byX, Y and Z. X allows linkage of PCB to the bimolecular substrate throughthe reactive group X′. Examples of X′ include Cl,O—N-hydroxysuccinimidyl, OCH₂CN, OPhF₅, OPhCl₅, N₃. Y represents asubstitution pattern of a phenyl ring containing one or moresubstitutions such as nitro or alkoxyl. The functionality Z represents agroup that allows linkage of the cross-linker moiety to thephotoreactive moiety. The photoreactive moiety has the property thatupon illumination, it undergoes a photoreaction that results in cleavageof the PCB molecule from the substrate.

A lysine-tRNA is misaminoacylated with photocleavable biotin-lysine, orchemically modified to attach a photocleavable biotin amino acid. Themisaminoacylated tRNA is introduced into a cell-free proteinsynthesizing system and nascent proteins produced. The nascent proteinscan be separated from other components of the system bystreptavidin-coated magnetic beads using conventional methods which relyon the interaction of beads with a magnetic field. Alternatively,agarose beads coated with streptavidin, avidin and there derivatives beutilized. Nascent proteins are released then from beads by irradiationwith UV light of approximately 280 nm wavelength. Once a nascent proteinis released from by light it can be analyzed in solution (homogenousphase) or transferred to another surface such as nitrocellulose,polystyrene or glass for anlaysis (solid phase analysis) (non-specificbinding surface or chemically activated). In one embodiment whichinvolves solid phase analysis, neutravidin-coated agarose beads are usedto capture nascent proteins produced in a cell-free rabbit reticulocyteprotein synthesis system and the beads then separated from the synthesissystem by centrifugation and washing. The nascent protein is thentransferred to the surface of a microplate well by inserting the beadsdirectly into the well and illuminating thereby facilitating transfer tothe well surface.

In one experimental demonstration, nascent proteins (p53 andalpha-tubulin) were produced in a rabbit reticulocyte protein synthesissystem supplemented with elongator tRNA misaminoacylated with aphotocleavable biotin derivatized lysine. Without further processing,the nascent proteins were then specifically captured on NeutrAvidinbiotin-binding agarose beads. After washing, the bead suspensioncontaining the immobilized nascent protein was added directly to thewells of a high-protein-binding polystyrene micro-well plate. The UVrelease was performed directly in the wells of the plate therebyallowing subsequent and immediate non-specific adsorption of thereleased target protein onto the surface of the well. This approach,which is facilitated by photocleavable biotin, eliminates the need forstabilizers/additives (e.g proteins like albumin or non-ionicdetergents) normally required when handling small quantities of puresoluble target protein separately in tubes or vials. Elimination of suchstabilizers/additives facilitates non-specific immobilization of theisolated target proteins and direct transfer of the target protein fromthe beads to the well of the plate minimizes handling and non-specificlosses. Furthermore, this approach eliminates the need for plates coatedwith proteinaceous capture elements and therefore should provide certainadvantages (e.g. lower background/interference from capture elements inthe plate-based immunoassay). Following removal of the beads from thewells by brief washes (e.g. 1-4 washes or more), the plate is ready forimmunoassay. In this case, the nascent proteins contained a C-terminalHSV epitope tag and detection was achieved using an anti-HSV antibodyconjugated to an alkaline phosphatase reporter. Signal was generated bythe addition of a chemiluminescence-based alkaline phosphatase substrateand analyzed in a plate reader. Using a 10 μL equivalent of translatedprotein, this initial experiment produced signal to noise ratios ofapproximately 140:1 and 610:1 for alpha-tubulin and p53, respectively,relative to a blank cell-free translation lacking only the added DNAconstruct.

Many devices designed to detect proteins are based on the interaction ofa target protein with specific immobilized acceptor molecule. Suchdevices can also be used to detect nascent proteins once they containaffinity markers such as biodetectors based on sensing changes insurface plasmons, light scattering and electronic properties ofmaterials that are altered due to the interaction of the target moleculewith the immobilized acceptor group.

Nascent proteins, including those which do not contain affinity-typemarkers, may be isolated by more conventional isolation techniques. Someof the more useful isolation techniques which can be applied or combinedto isolate and purify nascent proteins include chemical extraction, suchas phenol or chloroform extract, dialysis, precipitation such asammonium sulfate cuts, electrophoresis, and chromatographic techniques.Chemical isolation techniques generally do not provide specificisolation of individual proteins, but are useful for removal of bulkquantities of non-proteinaceous material. Electrophoretic separationinvolves placing the translation mixture containing nascent proteinsinto wells of a gel which may be a denaturing or non-denaturingpolyacrylamide or agarose gel. Direct or pulsed current is applied tothe gel and the various components of the system separate according tomolecular size, configuration, charge or a combination of their physicalproperties. Once distinguished on the gel, the portion containing theisolated proteins removed and the nascent proteins purified from thegel. Methods for the purification of protein from acrylamide and agarosegels are known and commercially available.

Chromatographic techniques which are useful for the isolation andpurification of proteins include gel filtration, fast-pressure orhigh-pressure liquid chromatography, reverse-phase chromatography,affinity chromatography and ion exchange chromatography. Thesetechniques are very useful for isolation and purification of proteinsspecies containing selected markers.

Another embodiment of the invention is directed to the incorporation ofnon-native amino acids or amino acid derivatives with marker or affinityproperties at the amino-terminal residue of a nascent protein (FIG. 13).This can be accomplished by using the side chain of an amino acid or byderivatizing the terminal amino group of an amino acid. In either casethe resulting molecule is termed an amino acid derivative. Theamino-terminal residue of a protein is free and its derivatization wouldnot interfere with formation of the nascent polypeptide. The non-nativeamino acid or amino acid derivative is then used to misaminoacylate aninitiator tRNA which only recognizes the first AUG codon signaling theinitiation of protein synthesis. After introduction of thismisaminoacylated initiator tRNA into a protein synthesis system, markeris incorporated only at the amino terminal of the nascent protein. Theability to incorporate at the N-terminal residue can be important asthese nascent molecules are most likely to fold into nativeconformation. This can be useful in studies where detection or isolationof functional nascent proteins is desired.

Not all markers incorporated with misaminoacylated initiator tRNAs atthe amino-terminal residue of the nascent protein show the sameacceptance by the protein translational machinery. Furthermore, therange of incorporation of different markers can be more restrictivecompared to the use of non-initiator tRNAs such as lysyl-tRNA. Althoughthe factors influencing this discrimination between markers forincorporation by a misaminoacylated initiator tRNA are not fullyelucidated, one possibility is that the initiation factor (IF2) which isused for carrying formylmethionine-tRNA^(fmet) to ribosomes plays arole. A second possibility is that the interaction between the markerstructure and the ribosomes plays a role. For example, the marker,BODIPY-FL is accepted by the protein translational machinery to agreater extent than smaller fluorescent markers such as NBD. For thisreason, BODIPY-FL is a superior marker for use in the detection ofnascent protein when incorporated through initiator tRNAs.

A marker group can also be incorporated at the N terminal by using amutant tRNA which does not recognize the normal AUG start codon. In somecases this can lead to a higher extent of specific incorporation of themarker. For example, the mutant of initiator tRNA, where the anticodonhas been changed from CAU—>CUA (resulting in the change of initiatormethionine codon to amber stop codon) has shown to act as initiatorsuppressor tRNA (Varshney U, RajBhandary U L, Proc Natl Acad Sci USA1990 February; 87(4):1586-90; Initiation of protein synthesis from atermination codon). This tRNA initiates the protein synthesis of aparticular gene when the normal initiation codon, AUG is replaced by theamber codon UAG. Furthermore, initiation of protein synthesis with UAGand tRNA (fMet^(CUA)) was found to occur with glutamine and notmethionine. In order to use this tRNA to introduce a marker at the Nterminal of a nascent protein, this mutant tRNA can be enzymaticallyaminoacylated with glutamine and then modified with suitable marker.Alternatively, this tRNA could be chemically aminoacylated usingmodified amino acid (for example methionine-BODIPY). Since proteintranslation can only be initiated by this protein on messages containingUAG, all proteins will contain the marker at the N-terminal end of theprotein.

D. Mass Spectrometry

Mass spectrometry measures the mass of a molecule. The use of massspectrometry in biology is continuing to advance rapidly, findingapplications in diverse areas including the analysis of carbohydrates,proteins, nucleic acids and biomolecular complexes. For example, thedevelopment of matrix assisted laser desorption ionization (MALDI) massspectrometry (MS) has provided an important tool for the analysis ofbiomolecules, including proteins, oligonucleotides, and oligosacharrides[Karas, 1987 #6180; Hillenkamp, 1993 #6175]. This technique's successderives from its ability to determine the molecular weight of largebiomolecules and non-covalent complexes (>500,000 Da) with high accuracy(0.01%) and sensitivity (subfemtomole quantities). Thus far, it has beenfound applicable in diverse areas of biology and medicine including therapid sequencing of DNA, screening for bioactive peptides and analysisof membrane proteins.

Markers incorporated by misaminoacylated tRNAs into nascent proteins,especially at a specific position such at the N-terminal can be used forthe detection of nascent proteins by mass spectrometry. Without such amarker, it can be very difficult to detect a band due to a nascentprotein synthesized in the presence of a cellular or cell-free extractdue the presence of many other molecules of similar mass in the extract.For example, in some cases less than 0.01% of the total protein mass ofthe extract may comprise the nascent protein(s). Furthermore, moleculeswith similar molecular weight as the nascent protein may be present inthe mixture. Such molecules will overlap with peaks due to the nascentprotein. This problem is particularly severe if the nascent protein is atranscription or translation factor already present in the cell-free orcellular protein synthesis. The synthesis of additional amounts of thisprotein in the protein synthesis system would be difficult to detectusing known methods in mass spectrometry since peak intensities are notcorrelated in a linear manner with protein concentration.

Detection by mass spectrometry of a nascent protein produced in atranslation system is also very difficult if the mass of the nascentprotein produced is not known. This might situation might occur forexample if the nascent protein is translated from DNA where the exactsequence is not known. One such example is the translation of DNA fromindividuals which may have specific mutations in particular genes orgene fragments. In this case, the mutation can cause a change in theprotein sequence and even result in chain truncation if the mutationresults in a stop codon. The mass of nascent proteins produced in atranslation system might also not be known if DNA is derived fromunknown sources such as as colonies of bacteria which can containdifferent members of a gene library or fragments thereof.

In one embodiment of the invention, the incorporation of markers of aspecific mass (mass markers) into nascent proteins can be used toeliminate all of the above mentioned problems associated with theconventional mass spectrometric approach. First, a tRNA misaminoacylatedwith a marker of a known mass is added to the protein synthesis system.The synthesis system is then incubated to produce the nascent proteins.The mass spectrum of the protein synthesis system is then measured. Thepresence of the nascent protein can be directly detected by identifyingpeaks in the mass spectrum of the protein synthesis system whichcorrespond to the mass of the unmodified protein and a second band at ahigher mass which corresponds to the mass of the nascent protein plusthe modified amino acid containing the mass of the marker.

There are several steps that can be taken to optimize the efficientdetection of nascent proteins using this method. The mass of the markershould exceed the resolution of the mass spectrometer, so that theincreased in mass of the nascent protein can be resolved from theunmodified mass. For example, a marker with a mass exceeding 100 daltonscan be readily detected in proteins with total mass up to 100,000 usingboth matrix assisted laser desorption (MALDI) or electrospray ionization(ESI) techniques. The amount of misaminoacylated tRNA should be adjustedso that the incorporation of the mass marker occurs in approximately 50%of the total nascent protein produced. An initiator tRNA is preferablefor incorporation of the mass marker since it will only be incorporatedat the N-terminal of the nascent protein, thus avoiding the possibilitythat the nascent protein will contain multiple copies of the massmarker.

One example of this method is the incorporation of the marker BODIPY-FL,which has a mass of 282, into a nascent protein using a misaminoacylatedinitiator tRNA. Incorporation of this marker into a nascent proteinusing a misaminoacylated initiator tRNA causes a band to appear atapproximately 282 daltons above the normal band which appears for thenascent protein. Since the incorporation of the marker is less than oneper protein due to competition of non-misaminoacylated E. colitRNA^(fmet), a peak corresponding to the unmodified protein alsoappears. Identification of these two bands separated by the mass of themarker allows initial identification of the band due to the nascentprotein. Further verification of the band due to the nascent protein canbe made by adjusting the level of the misaminoacylated initiator tRNA inthe translation mixture. For example, if the misaminoacylated initiatortRNA is left out, than only a peak corresponding to the unmodifiedprotein appears in the mass spectrum of the protein synthesis system. Bycomparing the mass spectrum from the protein synthesis system containingand not containing the misaminocylated tRNA with the BODIPY-FL, thepresence of the nascent protein can be uniquely identified, even when aprotein with similar or identical mass is already present in the proteinsynthesis system.

For the purpose of mass spectrometric identification of nascentproteins, it is sometimes advantageous to utilize a photocleavablemarker. In this case, peaks due to nascent proteins in the mass spectrumcan be easily identified by measuring and comparing spectra from samplesof the protein synthesis system that have been exposed and not exposedto irradiation which photocleaves the marker. Those samples which arenot exposed to irradiation will exhibit bands corresponding the mass ofthe nascent protein which has the incorporated mass marker, whereasthose samples which are exposed to irradiation will exhibit bandscorresponding to the mass of the nascent proteins after removal of themass marker. This shift of specific bands in the mass spectrum due toirradiation provides a unique identifier of bands which are due to thenascent proteins in the protein synthesis system.

One example of this method involves the use of the photocleavablemarker, photocleavable biotin. When photocleavable biotin isincorporated into the test protein α-hemolysin, a toxin produced byStaphyloccus, by using the misaminoacylated E. coli initiatortRNA^(met), the mass spectrum exhibits two peaks corresponding to themass of the nascent protein 35,904 Da, and a second peak at 36,465 Dacorresponding to the mass of the nascent protein plus the mass ofphotocleavable biotin. After photocleavage of the marker by exposing thecell-free or cellular extract to UV light with a wavelength ofapproximately 365 nm for approximately 10 minutes, the two bands undergochanges in intensity due to cleavage of the marker from the nascentprotein. For example, in the case of a form of photocleavable biotincontaining a single spacer the change in the mass will be 561.57. Thesecharacteristic changes are then used to uniquely identify the peakscorresponding the nascent protein. In the case of MALDI massspectrometry, the probe laser pulses when adjusted to sufficientintensity can be used to accomplish photocleavage of photocleavablebiotin. In this case, changes can be conveniently measured during thecourse of the measurements, thereby facilitating detection of peaksassociated with the nascent protein. A similar approach can also be usedto identify more than one nascent protein of unknown mass in a cell orcell-free translation system.

Markers with affinity properties which are incorporated bymisaminoacylated tRNAs into nascent proteins can also be very useful forthe detection of such proteins by mass spectrometry. Such markers can beused to isolate nascent proteins from the rest of the cell-free orcellular translation system. In this case, the isolation of the nascentproteins from the rest of the cell-free mixture removes interferencefrom bands due to other molecules in the protein translation system. Anexample of this approach is the incorporation of photocleavable biotininto the N-terminal end of a nascent proteins using misaminoacylatedtRNA. When this marker is incorporated onto the N-terminal end of anascent protein using an E. coli tRNA^(met), it provides a convenientaffinity label which can be bound using streptavidin affinity media suchas streptavidin agarose. Once the nascent protein is separated by thismethod from the rest of the protein synthesis system, it can be releasedby UV-light and analyzed by mass spectrometry. In the case of MALDI massspectrometry, release of the nascent protein can most conveniently beaccomplished by using the UV-laser excitation pulses of the MALDIsystem. Alternatively, the sample can be irradiated prior to massspectrometric analysis in the case of MALDI or ESI mass spectrometry.

E. Electrophoresis

Another embodiment of the invention is directed to methods for detectingby electrophoresis the interaction of molecules or agents with nascentproteins which are translated in a translation system. This methodallows a large number of compounds or agents to be rapidly screened forpossible interaction with the expressed protein of specific genes, evenwhen the protein has not been isolated or its function identified. Italso allows a library of proteins expressed by a pool of genes to berapidly screened for interaction with compounds or agents without thenecessity of isolating these proteins or agents. The agents might bepart of a combinatorial library of compounds or present in a complexbiological mixture such as a natural sample. The agents might interactwith the nascent proteins by binding to them or to cause a change in thestructure of the nascent protein by chemical or enzymatic modification.

In addition to gel electrophoresis, which measures the electrophoreticmobility of proteins in gels such as polyacyrlamide gel, this method canbe performed using capillary electrophoresis. CE measures theelectrophoretic migration time of a protein which is proportional to thecharge-to-mass ratio of the molecule. One form of CE, sometimes termedaffinity capillary electrophoresis, has been found to be highlysensitive to interaction of proteins with other molecules includingsmall ligands as long as the binding produces a change in thecharge-to-mass ratio of the protein after the binding event. The highestsensitivity can be obtained if the protein is conjugated to a markerwith a specifically detectable electromagnetic spectral property such asa fluorescent dye. Detection of a peak in the electrophoresischromatogram is accomplished by laser induced emission of mainly visiblewavelengths. Examples of fluorescent dyes include fluorescein,rhodamine, Texas Red and BODIPY.

It is very difficult to detect a nascent protein synthesized in acellular or cell-free extract by CE without subsequent isolation andlabeling steps due the need for high sensitivity detection and thepresence of many other molecules of similar mass/charge ratio in theextract. For example, in typical cases less than 0.01% of the totalprotein mass of the extract may comprise the nascent protein(s). Othermolecules with similar electrophoretic migration times as the nascentprotein may be present in the mixture. Such molecules will overlap withpeaks due to the nascent protein.

It is also very difficult using conventional methods of CE to detect theinteraction of molecules with nascent proteins produced in a cell freeor cellular synthesis system. Affinity capillary electrophoresis hasbeen found to be sensitive to interaction of proteins with othermolecules including small ligands as long as the binding produces achange in the charge-to-mass ratio of the protein after the bindingevent. However, the selective addition of a marker such as a fluorescentdye to a nascent protein is not possible using conventional meansbecause most markers reagents will nonspecifically label other moleculesin the protein synthesis system besides the nascent proteins. In somecases, it may be possible utilize a specific substrate or ligand whichbinds only to the nascent protein. However this approach requires adetailed knowledge of the binding properties of the nascent protein andspecial design of a ligand with marker properties. The nascent proteinmay also be isolated from the protein synthesis system and thenselectively labeled with a detectable marker. However, this alsorequires the development of a procedure for isolation of the nascentprotein which can be time consuming and requires extensive knowledge ofproperties of the protein or protein engineering to incorporate anaffinity epitope. Even after a nascent protein has been isolated, it isoften difficult to uniformly label the protein with a marker so that thecharge/mass ratio of each labeled protein remains the same. In the mostadvantageous form of labeling, a highly fluorescent marker isincorporated at only one specific position in the protein thus avoidinga set of proteins with different electrophoretic mobilities.

The method of the invention also overcomes major problems associatedwith the rapid screening of samples for new therapeutic compounds usingcapillary electrophoresis (CE) such as described in U.S. Pat. No.5,783,397 (hereby incorporated by reference) when the target protein isa nascent protein expressed in a translation system. This includes theneed to uniformly label expressed target proteins in a translationsystem with markers for high sensitivity analysis by CE which normallyrequires lengthy isolation steps and special techniques for uniformlabeling.

The method can also be used in conjunction with expression cloningmethod for isolating novel cDNA clones such as described in U.S. Pat.No. 565,451, which is specifically incorporated by reference. Thispatent describes novel methods to identify cDNA clones by a) collectingpools of about 100 individual bacterial colonies; and b) expressingproteins encoded by the cDNAs in the pools in vitro. Proteins which canbe identified in this manner include but are not limited to nucleic acidbinding protein, cytoskeletal protein, growth factor, differentiationfactor, post-translationally modified protein, phosphorylated protein,proteolytically cleaved protein, glycosylated protein, subunit or amultiple component of a protein complex, enzyme, isoform of a knownprotein, mutant form of known protein. Importantly, the method includesas a crucial step identifying a desired protein from protein translationsystem. Two such methods described for identifying the protein involveradioactive labeling and chemical labeling. However, these steps can beextremely time-consuming and are not conducive to rapidly screening anextract for the desired protein.

The present invention avoids all of the problems discussed above. In oneembodiment of the invention a tRNA misaminoacylated with a detectablemarker is added to the protein synthesis system. The system is incubatedto incorporate the detectable marker into the nascent proteins. One ormore molecules (agents) are then combined with the nascent proteins(either before or after isolation) to allow agents to interact withnascent proteins. Aliquots of the mixture are then subjected toelectrophoresis. Nascent proteins which have interacted with the agentsare identified by detecting changes in the electrophoretic mobility ofnascent proteins with incorporated markers. In the case where the agentshave interacted with the nascent proteins, the proteins can be isolatedand subsequently subjected to further analysis. In cases where theagents have bound to the nascent proteins, the bound agents can beidentified by isolating the nascent proteins.

In one example of this method, the fluorescent marker BODIPY-FL is usedto misaminoacylate an E. coli initiator tRNA^(fmet) as previouslydescribed. The misaminoacylated tRNA is then added to a proteinsynthesis system and the system incubated to produce nascent proteincontaining the BODIPY-FL at the N-terminal. A specific compound whichmay bind to the nascent protein is then added to the protein synthesissystem at a specific concentration. An aliquot from the mixture is theninjected into an apparatus for capillary electrophoresis. Nascentproteins in the mixture are identified by detection of the fluorescencefrom the BODIPY-FL using exciting light from an Argon laser tuned to 488nm. Interaction of the specific compound is determined by comparing theelectrophoretic mobility measured of the nascent protein exposed to thespecific compound with a similar measurement of the nascent protein thathas not been exposed. The binding strength of the compound can then beascertained by altering the concentration of the specific compoundsadded to the protein synthesis system and measuring the change in therelative intensity of bands assigned to the uncomplexed and complexednascent protein.

The method is not limited to studying the interaction of one agent withone nascent protein translated in a protein translation system. Forexample, a library of compounds can be screened to identify those whichserve are ligands for specific target protein. In addition tointeractions which involve the binding of one or more agents to thenascent proteins interactions which result in a modification of thenascent protein including but not limited to phosphorylation,proteolysis, glycosylation, formation of a complex with other biologicalmolecules can be detected using the marker incorporated in the nascentproteins when combined with electrophoresis. For example, theinteraction of an antibody with the nascent proteins can be detected dueto a change in the effective electrophoretic mobility of the complexformed. A similar approach could be used to identify the presence of oneor more compounds in a complex mixture which bind to the nascentprotein. Such a mixture might constitute a library of compounds producedby combinatorial chemistry or compounds which might be, present in acomplex biological mixture such as natural samples which may containtherapeutic compounds.

F. Microscale Methods

While the present invention contemplates capillary electrophoresis (seeabove), other methods are also contemplated. In particular, microscalemethods can be employed in conjunction with the novel markers (e.g.BODIPY) and methods of the present invention. The methods are“microscale” in that the dimensions of the channels on the device (andthe corresponding fluid volumes) are very small (typically in thepicometer range). For example, channels are typically betweenapproximately 0.10 and 0.50 μm in depth and between approximately 5 and500 μm in width.

Although there are many formats, materials, and size scales forconstructing integrated fluidic systems, the present inventioncontemplates microfabricated devices as a solution to the manyinefficiencies of larger scale screening. Devices can be microfabricatedfrom a number of materials. Silicon is the material used for theconstruction of computing microprocessors and its fabricationtechnologies have developed at an unprecedented pace over the past 30years. The principal modem method for fabricating semiconductorintegrated circuits is the so-called planar process. The planar processrelies on the unique characteristics of silicon and comprises a complexsequence of manufacturing steps involving deposition, oxidation,photolithography, diffusion and/or ion implantation, and metallization,to fabricate a “layered” integrated circuit device in a siliconsubstrate. See e.g., W. Miller, U.S. Pat. No. 5,091,328, herebyincorporated by reference. While this technology was initially appliedto making microelectronic devices, the same techniques are currentlybeing used for micromechanical systems.

Continuous flow liquid transport has been described using a microfluidicdevice developed with silicon. See J. Pfahler et al., Sensors andActuators, A21-A23 (1990), pp. 431-434. Pumps have also been described,using external forces to create flow, based on micromachining ofsilicon. See H. T. G. Van Lintel et al., Sensors and Actuators15:153-167 (1988). SDS capillary gel electrophoresis of proteins inmicrofabricated channels has also been described. See Yao S, et al.,“SDS capillary gel electrophoresis of proteins in microfabricatedchannels,” PNAS 96:5372 (1999). Compared to more conventionaltwo-dimensional denaturing gel electrophoresis (which is generally timeconsuming and requires considerable amounts of sample), thismicrochannel-based separation technique was shown to be quick and offerhigh resolution.

As a mechanical building material, silicon has well-known fabricationcharacteristics. The economic attraction of silicon devices is thattheir associated micromachining technologies are, essentially,photographic reproduction techniques. In these processes, transparenttemplates or masks containing opaque designs are used to photodefineobjects on the surface of the silicon substrate. The patterns on thetemplates are generated with computer-aided design programs and candelineate structures with line-widths of less than one micron. Once atemplate is generated, it can be used almost indefinitely to produceidentical replicate structures. Consequently, even extremely complexmicromachines can be reproduced in mass quantities and at lowincremental unit cost—provided that all of the components are compatiblewith the silicon micromachining process. While the present inventioncontemplates other substrates, such as glass or quartz, for use inphotolithographic methods to construct microfabricated analysis devices,silicon is preferred because of the added advantage of allowing a largevariety of electronic components to be fabricated within the samestructure.

In one embodiment, the present invention contemplates siliconmicromachined components in an integrated analysis system. Sample (e.g.a test compound) and one or more reagents (e.g. a BODIPY labellednascent protein) are injected either continuously or in pulses into thedevice through entry ports and they are transported through channels toa reaction chamber, such as a thermally controlled reactor where mixingand reactions take place. The biochemical products can be then moveddown a new channel (or by an electrophoresis module, if desired) wheremigration data is collected by a detector and transmitted to a recordinginstrument. If desired, a polymer can be used in the channels to provideresolution by molecular sieving. The biochemical products can beisolated by diverting the flow to an external port for subsequentadditional analysis. Importantly, the fluidic and electronic componentsare designed to be fully compatible in function and construction withthe biological reactions and reagents. In this embodiment, potentialtest compounds can be rapidly screened for interaction with a labelednascent protein or multiple nascent proteins that are co-expressed in atranslation reaction system. In this manner the system can be used toscreen for interaction so as to identify useful drugs.

In another embodiment, one or more components of the protein synthesissystem are introduced into the device through entry ports and they aretransported through channels to a reaction chamber, such as a thermallycontrolled reactor, where the expression of the nascent protein whichcontains the marker such as BODIPY occurs. The labeled nascent proteincan than be mixed with one or more reagents (e.g. a test compound) thatare introduced into the device through entry ports. After the reactiontakes placed, the biochemical products can be then moved down a newchannel (or by an electrophoresis module, if desired) where migrationdata is collected by a detector and transmitted to a recordinginstrument. It is to be understood that components of the proteinsynthesis system which can be introduced into the device can includemisaminoacylated tRNAs, DNA, mRNA, amino acids and nucleotides. Thecomponents can be introduced either continuously or in discrete pulses.The DNA may also be produced within the micromachined device byenzymatic reactions such as the polymerase chain reaction as has beendescribed. See Kopp et al., “Chemical Amplification: Continuous Flow PCRon a Chip,” Science 280:1046 (1998).

In silicon micromachining, a simple technique to form closed channelsinvolves etching an open trough on the surface of a substrate and thenbonding a second, unetched substrate over the open channel. There are awide variety of isotropic and anisotropic etch reagents, either liquidor gaseous, that can produce channels with well-defined side walls anduniform etch depths. Since the paths of the channels are defined by thephoto-process mask, the complexity of channel patterns on the device isvirtually unlimited. Controlled etching can also produce sample entryholes that pass completely through the substrate, resulting in entryports on the outside surface of the device connected to channelstructures.

G. Multiple Misaminoacylated tRNAs

It may often be advantageous to incorporate more than one marker into asingle species of protein. This can be accomplished by using a singletRNA species such as a lysine tRNA misaminoacylated with both a markersuch as dansyllysine and a coupling agent such as biotin-lysine.Alternatively, different tRNAs which are each misaminoacylated withdifferent markers can also be utilized. For example, the coumarinderivative could be used to misaminoacylate a tryptophan tRNA and adansyl-lysine used to misaminoacylate a lysine tRNA.

One use of multiple misaminoacylated tRNAs is to study the expression ofproteins under the control of different genetic elements such asrepressors or activators, or promoters or operators. For example, thesynthesis of proteins at two different times in response to an internalor external agent could be distinguished by introducing misaminoacylatedtRNAs at different times into the cellular or cell-free proteinsynthesis system. A tRNA^(tyr) might be charged with marker A and atRNA^(lys) charged with marker B, yielding A-tRNA^(tyr) andB-tRNA^(lys), respectively. In this case, protein one under the controlof one promoter can be labeled by adding the A-tRNA^(tyr) to thereaction system. If a second misaminoacylated tRNA, B-tRNA^(lys) is thenadded and a second promoter for protein two activated, the nascentprotein produced will contain both label A and B. Additional markerscould also be added using additional tRNA molecules to further study theexpression of additional proteins. The detection and analysis ofmultiply labeled nascent proteins can be facilitated by using themulti-colored electrophoresis pattern reading system, described in U.S.Pat. No. 5,190,632, which is specifically incorporated by reference, orother multi-label reading systems such as those described in U.S. Pat.Nos. 5,069,769 and 5,137,609, which are both hereby specificallyincorporated by reference.

A second use of multiple misaminoacylated tRNAs is in the combinedisolation and detection of nascent proteins. For example, biotin-lysinemarker could be used to misaminoacylate one tRNA and a coumarin markerused to misaminoacylate a different tRNA. Magnetic particles coated withstreptavidin which binds the incorporated lysine-biotin would be used toisolate nascent proteins from the reaction mixture and the coumarinmarker used for detection and quantitation.

A schematic diagram of the basics of the above methods is shown in FIG.14. In a first step, the marker selected (M), which may have reporter(R) or affinity (A) properties, is chemically or enzymaticallymisaminoacylated to a single tRNA species or a mixture of differenttRNAs. Prior to protein synthesis, a predetermined amount of themisaminoacylated tRNA, charged with the fluorescent marker is mixed withthe cell-free protein synthesis reaction system at concentrationssufficient to allow the misaminoacylated tRNA to compete effectivelywith the corresponding tRNA. After an incubation of about 1-3 hours, thereaction mixture is analyzed using conventional polyacrylamide oragarose gel electrophoresis. After electrophoresis, the gel isilluminated by UV radiation. Bands due to the nascent protein exhibitdistinct fluorescence and can be easily and rapidly distinguished,either visually or photographically, from non-fluorescent bands ofpreexisting proteins. Nascent proteins can be isolated by excising thefluorescent band and electroeluting the protein from the extracted gelpieces. The quantities and molecular weights of the nascent proteins canbe determined by comparison of its fluorescence with the fluorescenceproduced by a set of proteins with known molecular weights and knownquantities. The results of the assay can be recorded and stored forfurther analysis using standard electronic imaging and photographic orspectroscopic methods.

H. Resulting Compositions

Another embodiment of the invention is directed to a compositioncomprising nascent proteins isolated or purified by conventional methodsafter translation in the presence of markers. Compositions can beutilized in manufacturing for the preparation of reagents such ascoatings for tissue culture products and in the pharmaceutical industry.

Incorporation of markers into nascent proteins utilized in manufacturingfacilitates analysis of the final manufactured product or process bydetection of marker. For example, nascent proteins produced may be usedas coatings for tissue culture products. The reproducibility of aparticular coating process could be accurately determined by detectingvariations of marker emissions over the surface of the coated product.In addition, non-toxic markers incorporated into proteins encompassedwithin a pharmaceutical preparation such as a hormone, steroid, immuneproduct or cytokine can be utilized to facilitate safe and economicalisolation of that protein preparation. Such products could be useddirectly without the need for removal of marker. When very lowconcentrations of marker are preferred, limiting amounts of markedproteins could be used to follow a protein through a purificationprocedure. Such proteins can be efficiently purified and the purity ofthe resulting composition accurately determined. In addition, thepresence of markers may facilitate study and analysis of pharmaceuticalcompositions in testing. For example, markers can be utilized todetermine serum half-life, optimum serum levels and the presence orabsence of break-down products of the composition in a patient.

Alternatively, nascent proteins may contain specific markers which serveas therapeutically useful compounds such as toxic substances. Theseproteins are administered to a patient and the therapeutic moietyreleased after proteins have identified and possibly bound to theirrespective targets. Release may be electrical stimulation, photochemicalcleavage or other means whereby the moiety is specifically deposited inthe area targeted by the nascent proteins. In addition, moieties such asmodified toxins may be utilized which become toxic only after releasefrom nascent proteins. Nascent protein may also serve as apharmaceutical carrier which bestows the incorporated marker with activetherapeutic function or prevents marker from breaking down in the bodyprior to its therapeutic or imaging action.

The incorporation of cleavable markers in nascent proteins furtherprovides a means for removal of the non-native portion of the marker tofacilitate isolation of the protein in a completely native form. Forexample, a cleavable affinity marker such as photocleavable biotinintroduced into a nascent protein facilitates economical isolation ofthe protein and allows for the removal of the marker for further use asa pharmaceutical composition.

Pharmaceutical compositions of proteins prepared by translation in thepresence of markers may further comprise a pharmaceutically acceptablecarrier such as, for example, water, oils, lipids, polysaccharides,glycerols, collagens or combinations of these carriers. Usefulimmunological compositions include immunologically active compositions,such as a vaccine, and pharmaceutically active compositions, such as atherapeutic or prophylactic drug which can be used for the treatment ofa disease or disorder in a human.

I. Kits

Another embodiment of the invention is directed to diagnostic kits oraids containing, preferably, a cell-free translation containing specificmisaminoacylated tRNAs which incorporate markers into nascent proteinscoded for by mRNA or genes, requiring coupled transcription-translationsystems, and are only detectably present in diseased biological samples.Such kits may be useful as a rapid means to screen humans or otheranimals for the presence of certain diseases or disorders. Diseaseswhich may be detected include infections, neoplasias and geneticdisorders. Biological samples most easily tested include samples ofblood, serum, tissue, urine or stool, prenatal samples, fetal cells,nasal cells or spinal fluid. In one example, misaminoacylate fmet-tRNAscould be used as a means to detect the presence of bacteria inbiological samples containing prokaryotic cells. Kits would containtranslation reagents necessary to synthesize protein plus tRNA moleculescharged with detectable non-radioactive markers. The addition of abiological sample containing the bacteria-specific genes would supplythe nucleic acid needed for translation. Bacteria from these sampleswould be selectively lysed using a bacteria directed toxin such asColicin E1 or some other bacteria-specific permeabilizing agent.Specific genes from bacterial DNA could also be amplified using specificoligonucleotide primers in conjunction with polymerase chain reaction(PCR), as described in U.S. Pat. No. 4,683,195, which is herebyspecifically incorporated by reference. Nascent proteins containingmarker would necessarily have been produced from bacteria. Utilizingadditional markers or additional types of detection kits, the specificbacterial infection may be identified.

The present invention also contemplates kits which permit the GFTTdescribed above. For example, the present invention contemplates kits todetect specific diseases such as familial adenomatous polyposis. Inabout 30 to 60% of cases of familial adenomatous polyposis, the diseasedtissues also contain chain terminated or truncated transcripts of theAPC gene (S. M. Powell et al., N. Engl. J. Med. 329:1982-87, 1993).Chain termination occurs when frameshift cause a stop codon such as UAG,UAA or UGA to appear in the reading frame which terminates translation.Using misaminoacylated tRNAs which code for suppressor tRNAs, suchtranscripts can be rapidly and directly detected in inexpensive kits.These kits would contain a translation system, charged suppressor tRNAscontaining detectable markers, for example photocleavablecoumarin-biotin, and appropriate buffers and reagents. Such a kit mightalso contain primers or “pre-primers,” the former comprising a promoter,RBS, start codon, a region coding an affinity tag and a regioncomplementary to the template, the latter comprising a promoter, RBS,start codon, and region coding an affinity tag—but lacking a regioncomplementary to the template. The pre-primer permits ligation of theregion complementary to the template (allowing for customization for thespecific template used). A biological sample, such as diseased cells,tissue or isolated DNA or mRNA or PCR products of the DNA, is added tothe system, the system is incubated and the products analyzed. Analysisand, if desired, isolation is facilitated by a marker such as coumarinor biotin which can be specifically detected by its fluorescence usingstreptavidin coupled to HRP. Such kits provide a rapid, sensitive andselective non-radioactive diagnostic assay for the presence or absenceof the disease.

J. Colorectal Cancer PTT Detection

The present invention contemplates the isolation, detection andidentification of expressed proteins having an altered primary aminoacid sequence. One example of an altered primary sequence is a proteinchain truncation. A protein chain truncation is most easily explained bya frameshift mutation that generates a stop codon (i.e., AUG) within theopen reading frame. The resulting translation of the mRNA from thismutated gene synthesizes a nonfunctional or malfunctional protein. Oneexample of such a truncated protein is derived from the APC gene, and isknown to be a diagnostic marker for colorectal cancer.

Many attempts have been reported to detect and analyze biologicalsamples using a noninvasive diagnostic marker of colorectal cancer.Currently, the most reliable method to identify and treat colorectalcancer requires a colonoscopy. While colonoscopy is not a high riskprocedure, except for the associated general anesthesia, it is expensiveand there is a serious problem regarding obtaining compliance for onetime or repeated testing due to the invasive nature of the examinationand the extensive bowel preparation required. One possible non-invasivesource of diagnostic markers is fecal matter.

The Protein Truncation Test (PTT) was first reported by Roest et al.,Protein Truncation Test (PTT) For Rapid Detection OfTranslation-Terminating Mutations. Hum Mol Genet 2:1719-1721 (1993), andapplied to the detection of truncating mutations in the APC gene byPowell et al., Molecular Diagnosis Of Familial Adenomatous Polyposis. NEngl J Med 329:1982-1987 (1993). In traditional PTT, the region of thegene to be analyzed is amplified by PCR (or RT-PCR for an mRNA template)using a primer pair that incorporates additional sequences into the PCRamplicons required for efficient cell-free translation. The amplifiedDNA is then added to a cell-free transcription-translation extract alongwith radioactive amino acids (³⁵S-methionine or ¹⁴C-leucine). Theexpressed protein is analyzed by SDS-PAGE and autoradiography. Chaintruncation mutations are detected by the presence of a lower molecularweight (increased mobility) species relative to the wild-type (WT)protein band. Non-radioactive Western blot-based PTT-methods utilizing acombination of N-terminal and C-terminal epitopes have also beenreported. Rowan et al., Introduction Of A myc Reporter Tag To ImproveThe Quality Of Mutation Detection Using The Protein Truncation Test. HumMutat 9:172-176 (1997); de Koning Gans et al., A Protein Truncation TestFor Emery-Dreifuss Muscular Dystrophy (EMU): Detection Of N-TerminalTruncating Mutations. Neuromuscul Disord 9:247-250 (1999); and Kahamnnet al., A Non-Radioactive Protein Truncation Test For The SensitiveDetection Of All Stop And Frameshift Mutations. Hum Mutat 19:165-172(2002). However, these approaches still involve lengthy steps ofSDS-PAGE, electroblotting and membrane-based immunoassay.

Capillary electrophoreses provides an alternative to traditionalSDS-PAGE gels. For example, a translation carried out in presence ofBODIPY-FL tRNA results in a nascent protein (WT or mutant) havingincorporated the BODIPY-FL. As with SDS-PAGE, a mutant proteinexpressing a premature termination codon, will have faster mobility whenusing CE (i.e., a truncated protein). Capillary electrophoresis analysisof these translation samples run under denaturing conditions will resultin normal (i.e., WT) and mutant signal patterns as shown in FIG. 56. Therelative mobility difference determines the exact location if the mutantrelative to the normal protein, and depends on the position of thetruncation mutation(s).

As an alternative to SDS-PAGE based PTT, the present inventioncontemplates a high throughput solid-phase protein truncation test(HTS-PTT) that is compatible with multi-well or microarray formats. Theschematics for one embodiment of the HTS-PTT are shown in FIG. 41.Amplified DNA corresponding to the region of interest in the target geneis first generated using PCR with primers that incorporate N- andC-terminal epitope tags as well as a T7 promoter, Kozak sequence andstart codon (ATG) in the amplicons. (see Example 29) The resultingamplified DNA is subsequently added to a cell-free protein expressionsystem. (see Example 30) The cell-free transcription-translationreaction mixture is also supplemented with various misaminoacylatedtRNAs carrying affinity and/or detection tags. As illustrated in FIG.41, the incorporated affinity tags (e.g., biotins) are used to capturethe translated protein from the cell-free expression mixture onto asolid surface. The N- and C-terminal epitope tags are used to comparethe total amount of target protein bound (N-terminal signal) versus thefraction that is truncated (i.e., lacks a C-terminal). In addition,optional incorporation of a fluorescent label allows non-isotopic,direct detection of WT and truncated bands following SDS-PAGE. Thisfeature is useful during initial assay development allowing the resultsof the HTS-PTT to be compared with the results from fluorescent-basedSDS-PAGE. In the case of a positive diagnostic test for a chaintruncating mutation, the approximate position of the mutation can bedetermined using the fluorescent label feature followed by local DNAsequencing to determine the exact position and nature of the mutation.

As an initial evaluation of HTS-PTT, an ELISA-based multi-well assay wasdeveloped to detect truncating mutations in a region of the APC gene(segment 3; amino acids 1098-1696) using genomic DNA as a PCR template.Extensive screening of various epitope tag sequences including His-6,c-myc, P53 (derived from the P53 sequence), FLAG, VSV-G, Fil-16 (filaminderived) and StrepTag was performed in order to determine which wereoptimal with respect to signal-to-noise ratio. Based on this, VSV-G anda P53-derived tag were chosen as the N- and C-terminal epitopes,respectively. The target protein was expressed using a cell-freetranscription-translation system in the presence of a misaminoacylatedtRNAs (biotin-lysyl-tRNA and/or BODIPY-FL-lysyl-tRNA) designed toincorporate lysine residues modified with biotin or a fluorophore(BODIPY-FL) at random lysine positions. In order to enhance throughput,the nascent APC segment 3 was selectively captured from the reactionmixture via the incorporated biotin onto a 96-well ELISA plate andsimultaneously treated with the appropriate antibodies in a single step.Furthermore, to increase accuracy, the N- and C-terminal epitope tagswere measured in the same well plate using differentially labeledantibodies (HRP and alkaline phosphatase (AP), respectively).

DNA derived from patients with familial adenomatous polyposis (FAP) aswell as cell-line DNA with known mutations in segment 3 was analyzed byHTS-PTT (FIG. 42). WT APC yielded N- and C-terminal signals which were400 and 100-fold larger, respectively, than the background. The C/Nterminal ratio of WT APC was standardized to 100 and all other samplesare expressed relative to the WT. For the case of cell-line DNA, whichcontains homozygous mutations at codons 1367, 1416 and 1338 (FIG. 42;C1-C3, respectively), the C/N terminal ratio is close to 0 as expected.Since the N-terminal signals in all cases were approximately 200-foldgreater than background, this indicates that significant chain truncatedfragment is expressed and captured but essentially none are full-length.The C/N terminal ratio of samples derived from individuals with FAP(FIG. 42; P1-P4) ranged from 37% to 47% relative to the WT. Thevariation in the C/N terminal ratios is likely to reflect differences intranslation efficiency of the WT and mutant transcripts, as well asdifferences in capture and epitope accessibility which may depend on theposition of the mutation.

Validation of the HTS-PTT results was performed using the incorporatedfluorescent BODIPY labels FIG. 43 shows results obtained fromfluorescent imaging after SDS-PAGE on a small aliquots (4 μl) of thesame translation mixtures used in FIG. 42. The WT sample produces a bandof expected molecular mass (˜70 kDa), while the mutant APC cell-linesexhibited bands at approximately 35, 40 and 32 kDa (C1-C3 respectively),in agreement with the position of the chain truncating mutations asdetermined by DNA sequencing. In contrast, samples derived fromindividuals diagnosed with FAP all exhibited two bands corresponding toone WT and one mutant allele as expected for a heterozygote (lanesP1-P4). Furthermore, the band corresponding to the truncated fragmentexhibited an apparent molecular mass which is predicted by direct DNAsequencing (FIG. 43 caption).

While heterozygous mutations in germ-line cells are expected to comprise50% of the total DNA in a sample, sporadic mutations are often presentin significantly lower abundance, such as the case of stool samples fromindividuals with colorectal cancer. Traverso et al., Detection Of APCMutations In Fecal DNA From Patients With Colorectal Tumors. N Engl J.Med 346:311-320 (2002); Deuter et al., Detection Of APC Mutations InStool DNA Of Patients With Colorectal Cancer By HD-PCR. Hum Mutat,11:84-89 (1998); and Doolittle et al., Detection Of The Mutated K-RasBiomarker In Colorectal Carcinoma. Exp Mol Pathol 70:289-301 (2001). Onerecent approach which can detect as low as 0.40% mutant DNA relative toWT, termed digital PTT, was utilized as part of a non-invasive assay forcolorectal tumors. A key feature of this approach is the serial dilutionof DNA prior to PCR amplification, so that each reaction contains nomore than 4 copies of the APC gene. Detection of a mutation thusrequires that the PTT assay have sensitivity sufficient to detect 1 outof 4 (25%) mutated copies of the gene. 144 individual cell-freetranslation reactions were performed for each patient sample and eachreaction then analyzed by SDS-PAGE and autoradiography. Traverso et al.(2002). However, it would be desirable to replace the radioactivegel-based analyses with HTS-PTT in order to more efficiently screen suchlarge numbers of samples per patient.

In an experiment designed to measure the sensitivity of HTS-PTT, variousamounts of amplified WT and mutant APC DNA (cell-line C3) were mixed andtranslated as described earlier. As expected, the C/N terminal ratiodecreased with increasing levels of mutant DNA (FIGS. 44 & 45). C/Nterminal ratios were 100±6 (WT) versus 70±4 (25% mutant mixture; 3 WT:1mutant) and 42±4 (50% mutant mixture; 1 WT:1 mutant). An unpairedtwo-tailed t-test shows that the difference in raw C/N terminal ratiosbetween the WT and WT:mutant mixtures is statistically significant withp values of 1×10⁻¹¹ for WT versus 50% mixture and 1×10⁻⁸ for WT versus25% mixture (n=7). These results indicated that HTS-PTT may be suitableto replace the radioactive gel-based analysis in the digital PTT.Specifically, the above results indicated that the C/N ratio for the 50%and 25% mutant mixture deviate slightly from expected values of 0.5 and0.75, respectively. This deviation may possibly be due to unequalbinding and N-terminal accessibility of the full-length and truncatedfragments.

Experiments were also carried out using mRNA isolated from cell line C3which was then amplified using RT-PCR. The results (FIG. 44, brokenline) are very similar to those obtained using DNA as starting material(C/N terminal ratios for mRNA based HTS-PTT were 100±5, 64±3, 44±3 forWT, 25% mutant mixture and 50% mutant mixture, respectively). Thisdemonstrates the suitability of the HTS-PTT for analyzing chaintruncating mutation using mRNA. However, it is noted that most clinicallaboratories normally avoid the use of mRNA for PTT analysis because ofproblems such as the process of nonsense mediated mRNA decay, can makedetection of the mutated allele difficult in some cases. Frieschmeyer etal., Nonsense-Mediated mRNA Decay In Health And Disease, Hum Mol Genet8:1893-1900 (1999).

Several improvements are envisioned, for the basic HTS-PTT approachpresented herein. The use of biotin-lysyl-tRNA to incorporate biotinaffinity tags at lysine residues would result in no capture if the chaintruncation occurs upstream of the first lysine. This problem and theoverall efficiency of capture can be improved if a tRNA mixturecontaining most, or all, of the normal cellular tRNAs ismisaminoacylated with a biotin-labeled amino acid (i.e., tRNA^(TOTAL)).This “total tRNA mixture” is then used instead of lysyl-tRNA, therebymaking the biotin incorporation less dependent on the amino acidsequence of the nascent protein. It may also be possible to incorporatean affinity tag uniquely at the first residue in the sequence, therebyensuring capture of any size truncated protein. This has been achievedfor the case of an E. coli expression system using a suppressorinitiator tRNA in conjunction with a nonsense codon for initiation.Because the HTS-PTT is not limited by the resolution of SDS-PAGE, it ispossible to reduce the number of cell-free reactions per patient sampleby translating larger segments of the target gene (or the whole geneitself). In fact, initial studies indicate that HTS-PTT analysis offragments of at least 140 kDa in size is possible. Finally, the HTS-PTTis not limited to a multi-well ELISA/chemiluminescence format. Forexample, a microarray format is possible where the target proteins arecaptured on NeutrAvidin™ coated glass slides and detected usingfluorescently labeled antibodies.

In contrast to traditional methods of PTT, the HTS-PTT described hereinis non-isotopic, rapid and amenable to automation. The high throughputcapabilities of the HTS-PTT should be useful in order to facilitatepopulation-wide colorectal cancer (CRC) screening and other diseasesthat have prevalent truncation mutations.

The present invention contemplates the isolation, detection andidentification of mutated genes by methods that do not require extensiveand expensive purification, isolation and sequencing procedures.Furthermore, the present invention contemplates the use of nucleic acidmaterial from any tissue or fluid sample, and is not restricted to fecalsamples. Specifically, sample DNA from a patient suspected of havingcancer is amplified by PCR using primers comprising sequences encoding aN-terminal and C-terminal epitope. The epitope-containing sample DNA isplaced in a translation system (i.e., resulting in the production ofmRNA followed by protein synthesis) containing at least onemisaminoacylated marker tRNA. The marker is inserted into the nascentpeptide for affinity capture following protein synthesis. It is notintended that the tRNA be limited to a single misaminoacylated tRNA(i.e., for example, lysine). The present invention contemplates themisaminoacylation of all amino acid tRNA's with a marker (i.e., the“total tRNA” embodiment or tRNA^(TOTAL)). This approach uniformly labelsany length of any nascent protein with the affinity marker. Importantly,even if an amino acid in the C-terminal or N-terminal epitope receives amarker, the expected 1% incorporation rate (i.e., due to a lowmisaminoacylated tRNA concentration) will not reduce the ability todetect the affected epitope.

The present invention identifies a gene mutation by the ratio ofdetected N-terminal and C-terminal epitopes present in the nascentproteins. The epitopes may be identified by detection withenzyme-conjugated antibodies.

One embodiment of the present invention contemplates an HTS-PTT testcombined with a DNA-based method of detecting specific mutations in oneor more genes that have been associated with the series of geneticchanges which result in neoplastic transformation of normal colonicepithelium to benign adenomas and subsequently to malignantadenocarcinomas (Seung Myung Dong et al., J. Natl. Cancer Inst.,93:858-865 (2001)). It is also advantageous to utilize DNA-based assayswhich are compatible with the HTS-PTT platform and can be easilyimplemented in a clinical laboratory. For example, the TaqMan® assay andInvader® assay can be implemented on a 96, 384 or 1536 wellluminescent/fluorescent reader to detect missense, deletion andinsertion mutations which commonly occur in many genes, including APC.Even in cases where a large panel of known mutations is screened usingDNA-based probes (specifically designed for those mutations) asignificant percentage (i.e., approximately 20%) of de novo mutationsare likely to appear in the APC gene and not be detected (Gavert et.al., Molecular Analysis Of The APC Gene In 71 Israeli Families: 17 NovelMutations., Hum Mutat 19(6):664 (2002). These mutations can be detectedusing HTS-PTT. In contrast, such a panel combined with PTT is likely todetect such new mutations in the APC gene.

K. p53 Variants

The present invention contemplates PCR-mediated incorporation of a p53epitope variant into a diagnostic protein.

In one embodiment, the present invention contemplates variants of thegeneral formula:

-   -   T F S D L [x] K L L, wherein [x] can be any amino acid other        than W (SEQ ID NO:51).        Examples of such variants include (but are not limited to):

T F S D L H K L L (SEQ ID NO:24) T F S D L Y K L L (SEQ ID NO:25) T F SD L G K L L (SEQ ID NO:26) T F S D L N K L L (SEQ ID NO:27) T F S D L FK L L (SEQ ID NO:28) T F S D L D K L L (SEQ ID NO:29) T F S D L T K L L(SEQ ID NO:30)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   [z]_(y) T F S D L [x] K L L, wherein [x] can be any amino acid        other than W, [z] can be any amino acid including but not        limited to the amino acids corresponding to the wild-type        sequence, and y is an integer between 1 and 10 (SEQ ID NO:52).        Examples of such variants include (but are not limited to):

E T F S D L H K L L (SEQ ID NO:31) Q E T F S D L H K L L (SEQ ID NO:32)S Q E T F S D L H K L L (SEQ ID NO:33) L S Q E T F S D L H K L L (SEQ IDNO:34)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   [z]_(y) T F S D L [x] K L L [z]_(y), wherein [x] can be any        amino acid other than W, [z] can be any amino acid including but        not limited to the amino acids corresponding to the wild-type        sequence, and y is an integer between 1 and 10 (SEQ ID NO:53).        Examples of such variants include (but are not limited to):

E T F S D L H K L L P (SEQ ID NO:35) Q E T F S D L H K L L P (SEQ IDNO:36) S Q E T F S D L H K L L P (SEQ ID NO:37) L S Q E T F S D L H K LL P E (SEQ ID NO:38)L. VSV-G Variants

The present invention contemplates PCR-mediated incorporation of aneleven amino acid VSV-G epitope (residues 497-506) and variants thereof,into a diagnostic protein. This particular epitope is known to bind bothmonovalent and polyclonal antibodies and affects intracellular transportto the cell membrane. Kries, T. E., Microinjected Antibodies Against TheCytoplasmic Domain Of Vesicular Stomatitis Virus Glycoprotein Block It'sTransport To The Cell Surface. EMBO J, 5(5):931-941 (1986). Theincorporation of this VSV-G epitope into amphotropic leukemia virusenvelope glycoprotein retained compatibility with envelope processing,transport and incorporation, although some temperature-sensitive mutantswere generated. Battini et al., Definition Of A 14-Amino Acid PeptideEssential For The Interaction Between The Murine Leukemia VirusAmphotropic Envelope Glycoprotein And Its Receptor. J. Virol.,72(1):428-435 (1998).

By “variants” it is meant that the sequence need not comprise the exactsequence; up to three (3) amino acid substitutions are contemplated. Forexample, Leu or Ser may be substituted for the Gly; Ser may besubstituted for the Leu; and Ser or Ala may be substituted for the T.

In one embodiment, the present invention contemplates the wild typesequence:

Y T D I E M N R L G K (SEQ ID NO:39)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   Y [x] D I E M N R L G K, wherein [x] can be S or A (SEQ ID        NO:54).

An example of such a variant includes, but is not limited to:

Y A D I E M N R L G K (SEQ ID NO:40)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   Y T D I E M N R [y] G K, wherein [y] can be S (SEQ ID NO:55).        An examples of such a variant includes, but is not limited to:

Y T D I E M N R S G K (SEQ ID NO:41)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   Y T D I E M N R L [z] K, wherein [z] can be S or L (SEQ ID        NO:56).

An examples of such a variant includes, but is not limited to:

Y T D I E M N R L S K (SEQ ID NO:42)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   Y [x] D I E M N R [y] G K, wherein [x] can be S or A and [y] can        be S (SEQ ID NO:57).

An examples of such a variant includes, but is not limited to:

Y S D I E M N R S G K (SEQ ID NO:43)In another embodiment, the present invention contemplates variants ofthe general formula:

-   -   Y [x] D I E M N R L [z] K, wherein [x] can be S or A and [z] can        be G or S (SEQ ID NO:58).

An examples of such a variant includes, but is not limited to:

Y A D I E M N R L L K (SEQ ID NO:44)In another embodiment, the present invention contemplates variants ofthe general formula;

-   -   Y T D I E M N R [y] [z] K, wherein [y] can be S and [z] can be L        or S (SEQ ID NO:59).

An examples of such a variant includes, but is not limited to:

Y T D I E M N R S S K (SEQ ID NO:45)In another embodiment, the present invention contemplates variants ofthe general formula;

-   -   Y [x] D I E M N R [y] [z] K, wherein [x] can be S or A; [y] can        be S and [z] can be G, L or S (SEQ ID NO:60).        An examples of such a variant includes, but is not limited to:

Y A D I E M N R S G K (SEQ ID NO:46)

One embodiment of the present invention contemplates that by using amicrofluidic platform the amount of the translation mixture used forHTS-PTT assay can be lowered further than that contemplated in Example43. Technically, the entire HTS-PTT assay can be performed usingdisposable micro-fluidic chip.

First, translation is carried out in a first nano-channel where the DNAsample and misaminoacylated tRNA is injected into translation extractand mixed in a microchamber. After a short incubation, the wholetranslation mixture is then transported through a capillary coated withstreptavidin where binding of the translation products will occur. Afterbinding, the capillary is washed and fluorescently labeled antibodysolution, for example, dual color N-terminal and C-terminal antibodies,are passed through this capillary to allow the antibody to bind to thestreptavidin-bound protein. Finally, the capillary is read forN-terminal and C-terminal signal using fluorescence based detection(LIF-based detector).

A similar approach can be performed using a system consisting of aplurality of small through holes which can be loaded with fluids with avolume less than 1 microliter and even more preferable less than 100 nl.One example is described in U.S. Pat. No. 6,387,331 Method and apparatusfor performing microassays. Issued May 14, 2002, hereby incorporated byreference. In a preferred embodiment, a perforated platen is used havingsubstantially parallel planar surfaces for manipulating distinct liquidsamples, each sample having a volume less than 1 microliter, the platencomprising:

-   a. an inner layer of hydrophilic material;-   b. two outer layers of hydrophobic material coupled to opposite    sides of the inner layer in such a manner as to isolate the distinct    liquid samples from each other; and-   c. a two-dimensional array of through-holes for retaining the    distinct liquid samples, the through-holes each having a    characteristic cross-sectional dimension of between 100 and 400    micrometers, the through-holes spaced on a two-dimensional grid with    a center-to-center spacing between nearest neighbors of twice the    characteristic cross-sectional dimension or less, each through-hole    traversing the inner layer and the two outer layers in a direction    substantially perpendicular to the planar surfaces of the platen.

Using this system, a protein translation mixture such as a rabbitreticulocyte lysate can be loaded into the through-holes by capillaryaction. Loading may include drawing the said protein translation systemfrom a planar surface by capillary action. DNA is added to the proteintranslation mixture along with any desired addition components such asmisaminoacylated tRNA by loading a second set of samples in liquid forminto the through-holes of the second perforated platen; registering thethrough-holes of the first perforated platen with the through-holes ofthe second perforated platen; and combining the first set of sampleswith the second set of samples. Protein translation is carried out inthe through-holes by incubating the mixture using methods well-known inthe field. The translation mixture is then transported into thethrough-holes of a third perforated platen. In order to facilitatenascent protein capture, these through-holes (e.g. the surface) arecoated with streptavidin, avidin or its derivatives where binding of thetranslation products (e.g. nascent protein) occurs. After binding, thethrough-holes are washed and fluorescently labeled antibody solution,for example dual color N-terminal and C-terminal antibodies areintroduced into the through holes to allow the antibody to bind to thestreptavidin-bound protein. Finally, the through-holes are read forN-terminal and C-terminal signal using detection methods described inU.S. Pat. No. 6,387,331 employing an optical arrangement including adetector array for separately analyzing light emanating from each of theplurality of illuminated through-holes. In another embodiment, tRNAmisaminoacylated with photocleavable biotin is utilized to producenascent proteins which can be capture in streptavidin-coatedthrough-holes and then released for subsequent microanalysis of theproteins.

Another embodiment of the present invention contemplates a method forrapidly measuring cDNA library expression products. In this approach,the products were identified by Expression ELISA Assay.

Briefly, this method quickly assesses the product of the translationreaction in a high throughput manner. After the deconvolution of thecDNA pool, single colonies were grown and the DNA was isolated usingstandard mini-prep methods. The high-throughput ELISA-PTT assay was thenused to rapidly screen the DNA from several clones in order to determineif the DNA was expressed.

Specifically, FIG. 62 exemplifies the rapid assessment of thetranslation of individual clones from the cDNA library. The translationof individual DNA was carried out in rabbit reticulocyte lysate in thepresence of an equimolar mixture of BODIPY-FL-Lys-tRNA (or totalBODIPY-FL-Lys-tRNA) and Biotin-Lys-tRNA. After translation, the nascentprotein was transferred to neutravidin-coated plate and incubated for 30min. The plate was then washed extensively and the bound protein (i.e.,containing BODIPY) was detected using anti-BODIPY-FL antibody (MolecularProbes, Eugene, Oreg.) and HRP-conjugated anti-rabbit secondary antibody(Amersham Pharmacia Biotech, Piscataway, N.J.). Finally, the signal wasread using chemiluminescent ELISA reader Lumi-96 (Packard Biosciences,Meridian, Conn.).

Experimental

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention. In some ofthe examples below, particular reagents and methods were employed asfollows:

Reagents: tRNA^(fmet), aminoacyl-tRNA synthetase, amino acids, buffersalts, and RNase free water were purchased from Sigma (St. Louis, Mo.).Many of the fluorescent dyes were obtained from Molecular Probes(Eugene, Oreg.). The translation supplies including routine kits werepurchased from Promega (Madison, Wis.). Sephadex G-25 was fromAmersham-Pharmacia Biotech (Piscataway, N.J.). The in vitro translationkits and plasmid DNAs coding for CAT (PinPoint™) and Luciferase(PBESTluc™) were from Promega (Wisconsin-Madison, Wis.) while DHFRplasmid DNA (pQE16-DHFR) was obtained from Qiagen (Valencia, Calif.).The plasmid DNA for a-hemolysin, pT7-WT-H6-αHL was kindly supplied byProf. Hagan Bayley (Texas A &M University) and large scale preparationof α-HL DNA was carried out using Qiagen plasmid isolation kit. Thebacterioopsin plasmid DNA (pKKbop) was from the laboratory stock.

Preparation of FluoroTag tRNAs: The purified tRNA^(fmet) was firstaminoacylated with the methionine. In typical reaction, 1500 picomoles(˜1.0 OD₂₆₀) of tRNA was incubated for 45 min at 37° C. inaminoacylation mix using excess of aminoacyl tRNA-synthetases. Afterincubation, the mixture was neutralized by adding 0.1 volume of 3 Msodium acetate, pH 5.0 and subjected to chloroform:acid phenolextraction (1:1). Ethanol (2.5 volumes) was added to the aqueous phaseand the tRNA pellet obtained was dissolved in the water (25 μl). Thecoupling of NHS-derivatives of fluorescent molecules to the α-aminogroup of methionine was carried out in 50 mM sodium carbonate, pH 8.5 byincubating the aminoacylated tRNAf^(met) (25 μl) with fluorescentreagent (final concentration=2 mM) for 10 min at 0° C. and the reactionwas quenched by the addition of lysine (final concentration=100 mM). Themodified tRNA was precipitated with ethanol and passed through SephadexG-25 gel filtration column (0.5×5 cm) to remove any free fluorescentreagent, if present. The modified tRNA was stored frozen (−70° C.) insmall aliquots in order to avoid free-thaws. The modification extent ofthe aminoacylated-tRNA was assessed by acid-urea gel electrophoresis.This tRNA was found to stable at least for 6 month if stored properly.

Cell free synthesis of proteins and their detection: The in vitrotranslation reactions were typically carried out using E. coli T7transcription-translation system (Promega) with optimized premix. Thetypical translation reaction mixture (10 μl) contained 3 μl of extract,4 μl of premix, 1 μl of complete amino acid mix, 30 picomoles offluorescent-methionyl-tRNA and 0.5 μg of appropriate plasmid DNA. Theoptimized premix (1×) contains 57 mM HEPES, pH 8.2, 36 mM ammoniumacetate, 210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mMATP, 0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6mM cAMP and 16 mM magnesium acetate. The translation reaction wasallowed to proceed for 45 min at 37° C. For SDS-PAGE, 4-10 μl aliquot ofthe reaction mix was precipitated with 5-volume acetone and theprecipitated proteins were collected by centrifugation. The pellet wasdissolved in 1× loading buffer and subjected to SDS-PAGE after boilingfor 5 min. SDS-PAGE was carried out according to Laemmli and the gel wasscan using Molecular Dynamics FluorImager 595 using Argon laser asexcitation source. Alternatively, the nascent proteins in polyacrylamidegels were also detected using an UV-transilluminator and the photographswere carried out using Polaroid camera fitted with green filter (Tiffengreen #58, Polaroid DS34 camera filter kit).

For visualization of BODIPY-FL labeled protein, 488 nm as excitationsource was used along with a 530+/−30 narrow band excitation filter. Thegel was scanned using PMT voltage 1000 volts and either 100 or 200micron pixel size.

Enzyme/Protein activities: Biological activity of α-hemolysin wascarried out as follows. Briefly, various aliquots (0.5-2 μl) of in vitrotranslation reaction mixture were added to 500 μl of TBSA (Tris-bufferedsaline containing 1 mg/ml BSA, pH 7.5). To this, 25 μl of 10% solutionof rabbit red blood cells (rRBCs) was added and incubated at roomtemperature for 20 min. After incubation, the assay mix was centrifugedfor 1 min and the absorbance of supernatant was measured at 415 nm(release of hemoglobin). The equal amount of rRBCs incubated in 500 μlof TBSA is taken as control while rRBCs incubated with 500 μl of wateras taken 100% lysis. The DHFR activity was measuredspectrophotometrically. Luciferase activity was determined usingluciferase assay system (Promega) and luminescence was measures usingPackard Lumi-96 luminometer.

Purification of α-HL and measurement BODIPY-FL incorporation into α-HL:The translation of plasmid coding for α-HL (His₆) was carried out at 100μl scale and the α-HL produced was purified using Talon-Sepharose(ClonTech) according manufacturer instructions. The fluorescenceincorporated into α-HL was then measured on Molecular DynamicsFluorImager along with the several known concentration of free BODIPY-FL(used as standard). The amount of protein in the same sample wasmeasured using a standard Bradford assay using Pierce Protein Assay kit(Pierce, Rockford, Ill.).

FLAG Capture Assay

Biotinylation of FLAG Antibody

A 4.4 mg/mL stock of FLAG M2 monoclonal antibody (SIGMA Chemical, St.Louis, Mo.) is diluted with equal volume of 100 mM sodium bicarbonate(˜15 mM final antibody concentration). Subsequently, NHS-LC-Biotin(Pierce Chemical, Rockford, Ill.) is added from a 2 mM stock (in DMF) toa final 150 mM. The reaction is incubated for 2 hours on ice. Themixture is then clarified by centrifugation in a microcentrifuge (14,000R.P.M.) for 2.5 minutes. Unreacted labeling reagent is removed by gelfiltration chromatography.

Preparation of FLAG Antibody Coated ELISA Plates

NeutrAvidin™ biotin binding protein (Pierce Chemical, Rockford, Ill.) isdiluted to a final concentration of 50 mg/mL in 100 mM sodiumbicarbonate and used to coat Microlite (2+ white opaque 96-well ELISAplates (Dynex Technologies, Chantilly, Va.). Plates are washed withTBS-T and coated using a solution of 5 mg/mL biotinylated FLAG M2antibody in TBS-T. Plates are washed with TBS-T and blocked inTranslation Dilution Buffer (TDB) [4.5% Teleostean Gelatin, 2% non-fatmilk powder, 10 mM EDTA, 0.1% Tween-20, 1.25 mg/mL pre-immune mouse IgG,2.5 mM d-biotin, in TBS, pH 7.5.].

Binding and Detection of Target Protein

Triple-epitope-tagged target proteins produced by in vitro translationusing rabbit reticulocyte extract are diluted 1/25- 1/75 in TDB andadded to the antibody coated ELISA plates. Following capture of thetarget protein, plates are washed with TBS-T. Detection of c-myc isperformed using a polyclonal antibody (Santa Cruz Biotechnology, SantaCruz, Calif.) followed by a peroxidase labeled secondary antibody,whereas detection of the His₆ tag is achieved with a peroxidase labelednickel chelate-based probe (India(His Probe-HRP, Pierce, Rockford,Ill.). Antibodies are diluted in TDB and the India (His Probe-HRP isdiluted in TBS-T supplemented with 5 mg/mL pre-immune mouse IgG. In allcases, signal is generated using a chemiluminescent substrate system.

His-Tag Metal Affinity Capture ELISA Assay

Binding and Detection of Target Protein

Triple-epitope-tagged target proteins produced by in vitro translationusing rabbit reticulocyte extract are diluted 1/25- 1/75in 1% BSA/TBS-Tand added to nickel chelate coated ELISA plates (Pierce Chemical,Rockford, Ill.). Following capture of the target protein, plates arewashed with TBS-T and blocked with 1% BSA/TBS-T. Detection of epitopetags on the bound target protein is achieved using a monoclonal FLAG M2antibody (SIGMA Chemical, St. Louis, Mo.) or a polyclonal c-myc antibody(Santa Cruz Biotechnology, Santa Cruz, Calif.) in conjunction with theappropriate peroxidase labeled secondary antibody. Detection of biotinincorporated into the target protein via Biotin-lysyl-tRNA^(lys) isachieved using NeutrAvidin™ biotin binding protein conjugated toperoxidase (Pierce Chemical, Rockford, Ill.). The NeutrAvidin™ conjugateand all antibodies are diluted in 1% BSA/TBS-T. In all cases, signal isgenerated using a chemiluminescent substrate system.

EXAMPLE 1 Preparation of Markers

Synthesis of Coumarin Amino Acid: 4-(Bromomethyl)-7-methoxy coumarin(FIG. 15, compound 1; 6.18 mmole) and diethylacetamidomalonate (FIG. 15,compound 2; 6.18 mmole) were added to a solution of sodium ethoxide inabsolute ethanol and the mixture refluxed for 4 hours. The intermediateobtained (FIG. 15, compound 3) after neutralization of the reactionmixture and chloroform extraction was further purified bycrystallization from methanolic solution. This intermediate wasdissolved in a mixture of acetone and HCl (1:1) and refluxed for onehour. The reaction mixture was evaporated to dryness, and the amino acidhydrochloride precipitated using acetone. This hydrochloride wasconverted to free amino acid (FIG. 15, compound 4) by dissolving in 50%ethanol and adding pyridine to pH 4-5. The proton (¹H) NMR spectrum ofthe free amino acid was as follows: (m.p. 274-276° C., decomp.) —OCH₃ (δ3.85 s, 3H), —CH₂-(δ 3.5 d, 2H), α-CH— (δ 2.9 t, 1H), CH—CO (δ 6.25 s,1H), ring H (δ 7.05 s, 1H), (δ 7.8 d, 2H).

Synthesis of Fmoc derivative of coumarin: Coumarin amino acid (1.14mmol) was reacted with Fluorenylmethyloxycarbonyl N-hydroxysuccininmidylester (Fmoc-NHS ester) 1.08 mmol) in the presence of 1.14 mmol oftriethylamine for 30 minutes at room temperature. The reaction mixturewas acidified and the precipitate washed with 1 N HCl and dried. The NMRspectrum of the free amino acid was as follows: (MP 223-225° C.) —OCH₃(δ 3.85 s, 3H), —CH₂Br (δ 3.5 broad singlet, 2H), α-CH— (δ 3.0 t, 1H),CH—CO (δ 6.22 m, 1H), ring H (δ 7.05 s, 1H), (δ 7.8 d, 2H), fluorene HCH₂—CH (δ 4.2 m, 2H), CH₂—CH (δ 4.25 m, 1H), aromatic regions showedcharacteristic multiplets.

Synthesis of PCB: Photocleavable biotin was synthesized as describedbelow. 2-bromo, 2′-nitroacetophenone (FIG. 15, compound 5) was convertedfirst into its hexamethyltetraamommonium salt which was decomposed toobtain 2-amino, 2′-nitroacetophenone (FIG. 15, compound 6). BiotinN-hydroxysuccinimidyl ester (FIG. 15, compound 7; Sigma Chemical; St.Louis, Mo.) was reacted with a 6-aminocaproic acid (FIG. 15, compound 8)to obtain the corresponding acid (FIG. 15, compound 9). This acid wascoupled with the 2-amino, 2′-nitroacetophenone using DCC to obtain theketone (FIG. 15, compound 10). The ketone was reduced using sodiumborohydride to obtain the alcohol (FIG. 15, compound 11) which wasfurther converted into its chloroformate derivative (FIG. 15, compound12). The proton NMR spectrum of the derivative (compound 12) was asfollows: (δ 1.3 m, 3H), (δ 1.4 m, 2H), (δ 1.5 m, 5H) (δ 1.62 m, 1H), (δ2.1 t, 2H) (δ 2.4 t, 2H), (δ 2.6 d, 1H), (δ 2.8 m, 1H), (δ 3.0 t, 1H),(δ 3.1 m 1H), (δ 4.15 qt, 1H) (δ 4.42 qt, 1H), (δ 5.8 t, 1H), (δ 6.25 s,1H), (δ 6.45 s, 1H), (δ 7.5 t, 1H), (δ 7.75 m, 4H), (δ 7.9 d, 1H).

EXAMPLE 2 Misaminoacylation of tRNA

The general strategy used for generating misaminoacylated tRNA is shownin FIG. 16 and involved truncation of tRNA molecules, dinucleotidesynthesis (FIG. 17), aminoacylation of the dinucleotide (FIG. 18) andligase mediated coupling.

a) Truncated tRNA molecules were generated by periodate degradation inthe presence of lysine and alkaline phosphatase basically as describedby Neu and Heppel (J. Biol. Chem. 239:2927-34, 1964). Briefly, 4 mmolesof uncharged E. coli tRNA^(Lys) molecules (Sigma Chemical; St. Louis,Mo.) were truncated with two successive treatments of 50 mM sodiummetaperiodate and 0.5 M lysine, pH 9.0, at 60° C. for 30 minutes in atotal volume of 50 μl. Reaction conditions were always above 50° C. andutilized a 10-fold excess of metaperiodate. Excess periodate wasdestroyed treatment with 5 μl of 1M glycerol. The pH of the solution wasadjusted to 8.5 by adding 15 μl of Tris-HCl to a final concentration of0.1 M. The reaction volume was increased to 150 μl by adding 100 μl ofwater. Alkaline phosphatase (15 μl, 30 units) was added and the reactionmixture incubated again at 60° C. for two hours. Incubation was followedby ethanol precipitation of total tRNA, ethanol washing, drying thepellet and dissolving the pellet in 20 μl water. This process wasrepeated twice to obtain the truncated tRNA.

b) Dinucleotide synthesis was carried out basically as performed byHudson (J. Org. Chem. 53:617-24, 1988), and can be described as a threestep process, deoxycytidine protection, adenosine protection anddinucleotide synthesis.

Deoxycytidine protection: All reaction were conducted at roomtemperature unless otherwise indicated. First, the 5′ and 3′ hydroxylgroups of deoxycytidine were protected by reacting with 4.1 equivalentsof trimethylsilyl chloride for 2 hours with constant stirring. Exocyclicamine function was protected by reacting it with 1.1 equivalents ofFmoc-Cl for 3 hours. Deprotection of the 5′ and 3′ hydroxyl wasaccomplished by the addition of 0.05 equivalents of KF and incubationfor 30 minutes. The resulting product (FIG. 17, compound 19) wasproduced at an 87% yield. Phosphate groups were added by incubating thiscompound with 1 equivalent of bis-(2-chlorophenyl)phosphorochloridateand incubating the mixture for 2 hours at 0° C. The yield in this casewas 25-30%.

Adenosine protection: Trimethylsilyl chloride (4.1 equivalents) wasadded to adenosine residue and incubated for 2 hours, after which, 1.1equivalents of Fmoc-Cl introduced and incubation continued for 3 hours.The TMS groups were deprotected with 0.5 equivalents of fluoride ions asdescribed above. The Fmoc protected adenosine (compound 22) was obtainedin a 56% yield. To further protect the 2′-hydroxyl, compound 22 wasreacted with 1.1 equivalents of tetraisopropyl disiloxyl chloride(TIPDSCl₂) for 3 hours which produces compound 23 at a 68-70% yield. Thecompound was converted to compound 24 by incubation with 20 equivalentsof dihydropyran and 0.33 equivalents of p-toluenesulfonic acid indioxane for about 4-5 hours. This compound was directly convertedwithout isolation into compound 25 (FIG. 17) by the addition of 8equivalents of tetrabutyl ammonium fluoride in a mixture oftetrahydro-furan, pyridine and water.

Dinucleotide synthesis: The protected deoxycytidine, compound 20, andthe protected adenosine, compound 25 (FIG. 17), were coupled by theaddition of 1.1 equivalents of 2-chlorophenyl bis-(1-hydroxybenzotriazolyl)phosphate in tetrahydrofuran with constant stirring for30 minutes. This was followed by the addition of 1.3 equivalents ofprotected adenosine, compound 25, in the presence of N-methylimidazolefor 30 minutes. The coupling yield was about 70% and the proton NMRspectrum of the coupled product, compound 26 (FIG. 17), was as follows:(δ 8.76 m, 2H), (δ 8.0 m, 3H), (δ 7.8 m, 3H) (δ 7.6 m, 4H), (δ 7.5 m,3H), (δ 7.4 m, 18H), (δ 7.0 m, 2H), (δ 4.85 m, 14H), (δ 4.25 m, 1H); (δ3.6 m, 2H), (δ 3.2 m, 2H) (δ 2.9 m, 3H), (δ 2.6 m, 1H), (δ 2.0-1.2 m,7H).

c) Aminoacylation of the dinucleotide was accomplished by linking theprotected marker amino acid, Fmoc-coumarin, to the dinucleotide with anester linkage. First, the protected amino acid was activated with 6equivalents of benzotriazol-1-yl-oxy tris-(dimethylamino)phosphoniumhexafluoro phosphate and 60 equivalents of 1-hydroxybenzotriazole intetrahydrofuran. The mixture was incubated for 20 minutes withcontinuous stirring. This was followed with the addition of 1 equivalentof dinucleotide in 3 equivalents N-methylimidazole, and the reactioncontinued at room temperature for 2 hours. Deprotection was carried outby the addition of tetramethyl guanidine and 4-nitrobenzaldoxime, andcontinuous stirring for another 3 hours. The reaction was completed bythe addition of acetic acid and incubation, again with continuousstirring for 30 minutes at 0° C. which produced the aminoacylateddinucleotide (FIG. 18).

d) Ligation of the tRNA to the aminoacylated dinucleotide was performedbasically as described by T. G. Heckler et al. (Tetrahedron 40: 87-94,1984). Briefly, truncated tRNA molecules (8.6 O.D.₂₆₀ units/ml) andaminoacylated dinucleotides (4.6 O.D.₂₆₀ units/ml), were incubated with340 units/ml T4 RNA ligase for 16 hours at 4° C. The reaction bufferincluded 55 mM Na-Hepes, pH 7.5, 15 mM MgCl₂, 250 μM ATP, 20 μg/ml BSAand 10% DMSO. After incubation, the reaction mixture was diluted to afinal concentration of 50 mM NaOAc, pH 4.5, containing 10 mM MgCl₂. Theresulting mixture was applied to a DEAE-cellulose column (1 ml),equilibrated with 50 mM NaOAc, pH 4.5, 10 mM MgCl₂, at 4° C. The columnwas washed with 0.25 mM NaCl to remove RNA ligase and other non-tRNAcomponents. The tRNA-containing factions were pooled and loaded onto aBD-cellulose column at 4° C., that had been equilibrated with 50 mMNaOAc, pH 4.5, 10 mM MgCl₂, and 1.0 M NaCl. Unreacted tRNA was removedby washes with 10 ml of the same buffer. Pure misaminoacylated tRNA wasobtained by eluting the column with buffer containing 25% ethanol.

EXAMPLE 3 Preparation of Extract and Template

Preparation of extract: Wheat germ embryo extract was prepared byfloatation of wheat germs to enrich for embryos using a mixture ofcyclohexane and carbon tetrachloride (1:6), followed by drying overnight(about 14 hours). Floated wheat germ embryos (5 g) were ground in amortar with 5 grams of powdered glass to obtain a fine powder.Extraction medium (Buffer I: 10 mM trisacetate buffer, pH 7.6, 1 nMmagnesium acetate, 90 mM potassium acetate, and 1 mM DTT) was added tosmall portions until a smooth paste was obtained. The homogenatecontaining disrupted embryos and 25 ml of extraction medium wascentrifuged twice at 23,000×g. The extract was applied to a SephadexG-25 fine column and eluted in Buffer II (10 mM trisacetate buffer, pH7.6, 3 mM magnesium acetate, 50 mM potassium acetate, and 1 mM DTT). Abright yellow band migrating in void volume and was collected (S-23) asone ml fractions which were frozen in liquid nitrogen.

Preparation of template: Template DNA was prepared by linearizingplasmid pSP72-bop with EcoRI. Restricted linear template DNA waspurified by phenol extraction and DNA precipitation. Large scale mRNAsynthesis was carried out by in vitro transcription using theSP6-ribomax system (Promega; Madison, Wis.). Purified mRNA was denaturedat 67° C. for 10 minutes immediately prior to use.

EXAMPLE 4 Cell-Free Translation Reactions

The incorporation mixture (100 μl) contained 50 μl of S-23 extract, 5 mMmagnesium acetate, 5 mM Tris-acetate, pH 7.6, 20 mM Hepes-KOH buffer, pH7.5; 100 mM potassium acetate, 0.5 mM DTT, 0.375 mM GTP, 2.5 mM ATP, 10mM creatine phosphate, 60 μg/ml creatine kinase, and 100 μg/ml mRNAcontaining the genetic sequence which codes for bacterioopsin.Misaminoacylated PCB-lysine or coumarin amino acid-tRNA^(lys) moleculeswere added at 170 μg/ml and concentrations of magnesium ions and ATPwere optimized. The mixture was incubated at 25° C. for one hour.

EXAMPLE 5 Isolation of Nascent Proteins Containing PCB-Lysine

Streptavidin coated magnetic Dynabeads M-280 (Dynal; Oslo, Norway),having a binding capacity of 10 μg of biotinylated protein per mg ofbead. Beads at concentrations of 2 mg/ml, were washed at least 3 timesto remove stabilizing BSA. The translation mixture containing PCB-lysineincorporated into nascent protein was mixed with streptavidin coatedbeads and incubated at room temperature for 30 minutes. A magnetic fieldwas applied using a magnetic particle concentrator (MPC) (Dynal; Oslo,Norway) for 0.5-1.0 minute and the supernatant removed with pipettes.The reaction mixture was washed 3 times and the magnetic beads suspendedin 50 μl of water.

Photolysis was carried out in a quartz cuvette using a Black-Raylongwave UV lamp, Model B-100 (UV Products, Inc.; San Gabriel, Calif.).The emission peak intensity was approximately 1100 μW/cm² at 365 nm.Magnetic capture was repeated to remove the beads. Nascent proteinsobtained were quantitated and yields estimated at 70-95%.

EXAMPLE 6 The Lower Limit of Detection Using Fluorescence

Bovine serum albumin (BSA), suspended at 0.25 mg/ml in borate buffer, pH8.0, was combined with a 25 fold molar excess fluorescamine (SigmaChemical; St. Louis, Mo.) at 50 mg/ml to produce a modified, fluorescentBSA. Various amounts of modified protein (1 ng, 5 ng, 10 ng, 25 ng, 50ng, 75 ng, 100 ng, 150 ng, 200 ng) were suspended in loading buffer(bromophenol blue, glycerol, 2-mercaptoethanol, Tris-HCl, pH 6.8, SDS),and added to individual wells of a 1.5 mm thick, 12% polyacrylamide gelwith a 3% stacker. The water cooled gel was electrophoresed for 4 hoursat 50 volts. After electrophoresis, the gel was removed from theelectrophoresis apparatus, placed on a UV transilluminator andphotographed with polaroid Type 667 film using an exposure time of 10seconds. The lowest limit of detection observed under theses conditionswas 10 ng. These results indicate that using equipment found in atypical molecular biology lab, fluorescently labeled proteins can bedetected at ng quantities. Using even more sophisticated detectionprocedures and devices the level of detection can be increased evenfurther.

EXAMPLE 7 Nascent Proteins Containing Coumarin-Amino Acid

Cell-free translation is performed as described using charged tRNA^(lys)molecules misaminoacylated with lysine coupled to a benzopyrenefluorophore moiety and human γ-interferon mRNA which contains 21 codonsfor lysine. Samples of the mixture are supplemented with buffercontaining bromophenol blue, glycerol, 2-mercaptoethanol, Tris-HCl, pH6.8, and SDS, and directly applied to a 12% poly-acrylamide gel (3%stacker) along with a set of molecular weight markers. Electrophoresisis performed for 3 hours at 50 volts. The gel is removed from theelectrophoresis apparatus and photographed under UV light. Bands offluorescently labeled interferon protein are specifically detected at amolecular weight of about 25 KDa. No other significant fluorescentactivity is observed on the gel. Free misaminoacylated tRNA moleculesmay be electrophoresed off of the gel and not specifically detected.

EXAMPLE 8 In Vivo Half-life of a Pharmaceutical Composition

Cell-free translation reactions are performed by mixing 10 μl ofPCB-coumarin amino acid-tRNA^(leu), prepared by chemicalmisaminoacylation as described above and suspended in TE at 1.7 mg/ml),50 μl of S-23 extract, 10 μl water and 10 μl of a solution of 50 mMmagnesium acetate, 50 mM Tris-acetate, pH 7.6, 200 mM Hepes-KOH buffer,pH 7.5; 1 M potassium acetate, 5 mM DTT, 3.75 mM GTP, 25 mM ATP, 100 mMcreatine phosphate and 600 μg/ml creatine kinase. This mixture is kepton ice until the addition of 20 μl of 500 μg/ml human IL-2 mRNA(containing 26 leucine codons) transcribed and isolated from recombinantIL-2 cDNA. The mixture is incubated at 25° C. for one hour and placed onice. 100 μl of streptavidin coated magnetic Dynabeads (2 mg/ml) areadded to the mixture which is placed at room temperature for 30 minutes.After incubation, the mixture is centrifuged for 5 minutes in amicrofuge at 3,000×g or, a magnetic field is applied to the solutionusing a MPC. Supernatant is removed and the procedure repeated threetimes with TE. The final washed pellet is resuspended in 50 μl of 50 mMTris-HCl, pH 7.5 and transferred to a quartz cuvette. UV light from aBlack-Ray longwave UV lamp is applied to the suspension forapproximately 1 second. A magnetic field is applied to the solution witha MPC for 1.0 minute and the supernatant removed with a pipette. Thesupernatant is sterile filtered and mixed with equal volumes of sterilebuffer containing 50% glycerol, 1.8% NaCl and 25 mM sodium bicarbonate.Protein concentration is determined by measuring the O.D.₂₆₀.

0.25 ml of the resulting composition is injected i.v. into the tail veinof 2 Balb/c mice at concentrations of 1 mg/ml. Two control mice are alsoinjected with a comparable volume of buffer. At various time points (0,5 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours and 6 hours), 100μl serum samples are obtained from foot pads and added to 400 μl of 0.9%saline. Serum sample are added to a solution of dynabeads (2 mg/ml)coated with anti-coumarin antibody and incubated at room temperature for30 minutes. A magnetic field is applied to the solution with a MPC for 1minute and the supernatant removed with a pipette. Fluorescence at 470nm is measured and the samples treated with monoclonal antibody specificfor rat IL-2 protein. IL-2 protein content is quantitated for eachsample and equated with the amount of fluorescence detected. From theresults obtained, in vivo IL-2 half-life is accurately determined.

EXAMPLE 9 Incorporation of Various Fluorophores into α-Hemolysin

E. coli tRNA^(fmet) was first quantitatively aminoacylated withmethionine and the α-amino group was specifically modified usingNHS-derivatives of several fluorophores. The list of fluorescentreporter molecules (fluorophores) tested and their properties are givenin Table 2. Under the modification conditions, the modifiedMet-tRNA^(fmet) is found to be stable as assessed by acid-urea gel.Since all the fluorescent molecules tested have different opticalproperties (excitation and emission), we have determined their relativefluorescence intensity under the condition which were used for thequantitation of gels containing nascent protein.

Fluorescent detection of nascent protein was first evaluated usingα-hemolysin (α-HL) as a model protein (with C-terminal His₆-tag). α-HLis a relatively small protein (32 kDa) and could be produced efficientlyin in vitro translation. In addition, its activity can be measureddirectly in the protein translation mixture using a rabbit red bloodcell hemolysis assay. In vitro translation of α-HL was carried out usingan E. coli T7 S30 transcription/translation extract (Promega Corp.,Madison, Wis.) in the presence of several different modifiedmethionyl-tRNA^(fmet) as described above. After the reaction, an aliquot(3-5 μl) was subjected to SDS-PAGE analysis and the fluorescent bandswere detected and quantitated using a FluorImager F595 (MolecularDynamics, Sunnyvale, Calif.).

The data is presented in FIG. 20. Lane 1 is a no DNA control. Lane 2shows the results with BODIPY-FL-SSE. Lane 3 shows the results withBODIPY-FL-SE. Lane 4 shows the results with NBD (see Table 2 for thestructure). Lane 5 shows the results with Bodipy-TMR. Lane 6 shows theresults with BODIPY R6G. Lanes 7, 8, 9 and 10 show the results achievedwith FAM, SFX, PYMPO and TAMRA, respectively (see Table 2 forstructures).

The results clearly indicate the α-HL produced in presence ofBODIPY-FL-methionyl-tRNA^(fmet) (lanes 2 and 3) exhibited the highestfluorescence (all the data is normalized to the BODIPY-FL-SSE. The twodifferent BODIPY-FL reagents (BODIPY-FL sulfosuccinimidyl ester (SSE)and BODIPY-FL succinimidyl ester (SE)), differ only with respect tosolubility. The next best fluorophore evaluated,6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid,succinimidyl ester (TAMRA-X, SE), exhibited 35% of the fluorescence(corrected for relative fluorescence) of BODIPY-FL-SSE. Two other formsof BODIPY, BODIPY-TMR, SE(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoicacid, succinimidyl ester) and BODIPY-R6G, SE(4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid,succinimidyl ester) exhibited less than 3% of the fluorescence ofBODIPY-FL, SSE. Succinimidyl6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoate (NBD-X-SE), afluorescent molecule which has previously been incorporated into theneuorkinin-2 receptor exhibited only 6% of the BODIPY-FL-SSE. The twofluorescein analogs 5-(and-6)-carboxyfluorescein, succinimidyl-ester(FAM, SE) and 6-(fluorescein-5-(and-6)carboxamido)hexanoic acid,succinimidyl ester (SFX, SE) also showed very low fluorescence (8.4% and4.6%, respectively relative to BODIPY-FL).

EXAMPLE 10 Optimizing Incorporation

In order to optimize the amount of BODIPY-FL incorporated into nascentproteins, the translation reaction for α-HL was carried out in presenceof increasing amounts of BODIPY-FL-methionyl-tRNA^(fmet) ranging from3-60 picomoles per reaction. All reactions yielded similar amount ofα-HL as determined by hemolysis activity of rabbit red blood cellsindicating that the exogenously added BODIPY-FL-methionyl-tRNAe^(fmet)in this range did not inhibit protein synthesis. In contrast, theintensity of the fluorescent band corresponding to α-HL continued toincrease up to 30 picomoles BODIPY-FL-methionyl-tRNA per 10 μl reaction(data not shown). Increases above this level produced no furtherincrease in fluorescence, thus subsequent reactions were performed usingthis level of BODIPY-FL-methionyl-tRNA.

A second step used to optimize BODIPY-FL incorporation was based oneliminating N-formyl-tetrahydrofolate (fTHF) from the reaction mixture.In prokaryotes, N-formyl-tetrahydrofolate (fTHF) acts as a cofactor forthe enzyme methionyl-tRNA transformylase, which formylates the initiatortRNA after its aminoacylation with methionine. Protein synthesis is theninitiated using this modified tRNA (formyl-methionine-tRNA). Withoutlimiting the invention to any particular mechanism, it is believed thateliminating fTHF from the reaction mixture reduces the competition forinitiation of protein synthesis between this endogenous initiator tRNAand exogenously added modified-initiator RNA by preventing theformylation of endogenous initiator tRNA. This was confirmed bymeasuring fluorescence directly from SDS-PAGE for reactions for whichfTHF was present and absent from the reaction mixture. In the latercase, a 2-3 fold increase in fluorescence was found (data not shown).

EXAMPLE 11 Incorporation into other Proteins

In order to explore the general applicability of this approach,transcription/translation reactions with BODIPY-FL-methionyl-tRNA^(fmet)were carried out using various plasmid DNAs coding for dihydrofolatereductase (DHFR), luciferase, chloramphenicol acetyl-transferase (CAT)and bacteriorhodopsin (BR). BR was included because it representsmembrane proteins, which are typically very hydrophobic. An optimizedcoupled transcription/translation system was used along with freeBODIPY-FL and BODIPY-FL-methionyl-tRNA^(fmet) using the Talon metalchelate resin (ClonTech, Palo Alto, Calif.) in order to examineincorporation into other proteins. The results are shown in FIGS. 21A(visualization using laser based Molecular Dynamics FluorImager 595) and21B (visualization using a UV-transilluminator). Lane 1 is a no DNAcontrol. Lanes 2, 3, 4, 5 and 6 are hemolysin, DHFR, Luciferase, CAT andbacteriohodopsin, respectively.

Fluoresceht bands are observed using a fluorescence scanner for each ofthe proteins at positions corresponding to their relative molecular. Inthe case of luciferase, bands are observed which correspond to theexpected products of false initiation at internal methionines (PromegaTechnical Bulletin TB219). Bands corresponding to all of the proteinscould also be observed visually and recorded photographically using a UVtransilluminator (UVP TMW-20) combined with an emission filter thatallows light with λ>450 nm (FIG. 21B). Excitation in this case is likelyoccur in the UV absorbing band of BODIPY-FL which extends from 300-400nm.

The amount of BODIPY incorporation was then determined by measuring theamount of incorporated BODIPY-FL and protein present in the purifiedsample by comparison with solutions of different concentrations ofBODIPY-FL and using a Bradford protein assay, respectively. The averageof three such measurements yielded a molar ratio of 0.29+/−0.03%.However, the incorporation yield is likely to be higher sincefluorescence quenching of BODIPY-FL with protein residues such astryptophan and tyrosine may lower the fluorescence quantum yieldcompared to BODIPY-FL in aqueous solution.

The effects of the fluorescence labeling procedure on the activity ofthe nascent proteins synthesized was also evaluated. This is importantin cases where it is desirable to perform downstream functional analysissuch as in the case of in vitro expression cloning and other proteomicapplications of in vitro technology. Although it is possible for theN-terminal fluorescent label to alter the function of a protein, the lowmolar incorporation level (˜0.3%) should not significantly alter theoverall activity of the extract. This is confirmed by various enzymeassays and no significant difference is found for the activity measuredfor DHFR, α-HL and luciferase synthesized in the presence and absence ofthe BODIPY-FL-methionyl-tRNA^(fmet) (see Table 3).

EXAMPLE 12 Measuring the Sensitivity

In order to estimate the sensitivity of the method, various dilutions ofthe translation extract corresponding to 0.003-0.5 μl of the originalreaction mixture were analyzed by SDS-PAGE. As a control, extract from areaction performed without DNA was analyzed. As seen in FIG. 22B,fluorescence from α-HL bands corresponding to as small as 0.007 μl ofthe original reaction mixture were detectable. Based on the ourestimation of total nascent protein produced in the in vitro system,which ranged from 50-80 μg/ml, this corresponds to 0.35-0.5 nanograms ofα-hemolysin. This compares favorably with the sensitivity obtainableusing radioisotope labeling of nascent proteins where such a lowexpression of nascent protein may required longer exposure to X-ray filmwhich might result in serious background problem. It also exceeds thesensitivity of measuring proteins on gels currently with commerciallyavailable dyes such as Coomassie Blue (8-100 nanograms). Furtherimprovements in sensitivity are expected by increasing the level ofBODIPY-FL incorporation and by reducing background fluorescence, whichappears to be due to fluorescent impurities in the gel material, extractand modified tRNA added.

EXAMPLE 13 Synthesis as a Function of Time

The ability of the fluorescent labeling approach to monitor the nascentprotein synthesis as a function of time was also evaluated. For thispurpose, small aliquots of the α-HL transcription/translation mixture (4μl) were withdrawn at various times during the reaction and analyzed bySDS-PAGE. As seen in FIG. 22A, bands due to α-HL can clearly be detectedas early as 5 minutes after initiation of the incubation. Synthesis offluorescently labeled α-HL appears to saturate after 15 minutes oftranslation.

EXAMPLE 14 The Modifying Reagent

In the case of post-aminoacylation modifications used to form amisaminoacylated tRNA, an important factor is the modifying reagent usedto add the modification to the natural amino acid. For example, in thecase of the fluorophore BODIPY FL, there are two different commerciallyavailable BODIPY FL NHS reagents known as BODIPY-FL-SE and BODIPY-FL-SSE(Molecular Probes). Both reagents are based on N-hydroxysucinimide (NHS)as the leaving group. However, the two forms differ in aqueoussolubility due to the presence in one form (SSE) of a sulfonate (sulfo)group (see Table 2 for structures). In this example, optimized reactionsbased on standard biochemical procedures were performed aimed at addingthe BODIPY FL fluorophore to a purified tRNA^(fmet) which isaminoacylated with methionine using these two different reagents. Forthis purpose, first the tRNA^(fmet) was aminoacylated with themethionine. In typical reaction, 1500 picomoles (˜1.0 OD₂₆₀) of tRNA wasincubated for 45 min at 37° C. in aminoacylation mix using excess ofaminoacyl tRNA-synthetases. The aminoacylation mix consisted of 20 mMimidazole-HCl buffer, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 2 mM ATP and1600 units of aminoacyl tRNA-synthetase. The extent of aminoacylationwas determined by acid-urea gel as well as using ³⁵S-methionine. Afterincubation, the mixture was neutralized by adding 0.1 volume of 3 Msodium acetate, pH 5.0 and subjected to chloroform:acid phenol (pH 5.0)extraction (1:1). Ethanol (2.5 volumes) was added to the aqueous phaseand the tRNA pellet obtained was dissolved in water (37.5 (1) and usedfor modification.

A. Modification of Aminoacylated tRNA with BODIPY-FL-SSE

To the above aminoacylated-tRNA solution, 2.5 (1 of 1N NaHCO₃ was added(final conc. 50 mM, pH=8.5) followed by 10 (1 of 10 mM solution ofBODIPY-FL-SSE (Molecular Probes) in water. The mixture was incubated for10 min at 0° C. and the reaction was quenched by the addition of lysine(final concentration=100 mM). To the resulting solution 0.1 volume of 3M NaOAc, pH=5.0 was added and the modified tRNA was precipitated with 3volumes of ethanol. Precipitate was dissolved in 50 ml microliters ofwater and purified on Sephadex G-25 gel filtration column (0.5×5 cm) toremove any free fluorescent reagent, if present. The modified tRNA wasstored frozen (−70° C.) in small aliquots in order to avoid free-thaws.

B. Modification of Aminoacylated tRNA with BODIPY-FL-SE

To the above aminoacylated-tRNA solution, 2.5 (1 of 1N NaHCO₃ (finalconc. 50 mM, pH=8.5) and 20 (1 of DMSO was added followed by 10 (1 of 10mM solution of BODIPY-FL-SE (Molecular Probes) in DMSO. The mixture wasincubated for 10 min at 0° C. and the reaction was quenched by theaddition of lysine (final concentration=100 mM). To the resultingsolution 0.1 volume of 3 M NaOAc, pH =5.0 was added and the modifiedtRNA was precipitated with 3 volumes of ethanol. Precipitate wasdissolved in 50 ml of water and purified on Sephadex G-25 gel filtrationcolumn (0.5×5 cm) to remove any free fluorescent reagent, if present.The modified tRNA was stored frozen (−70° C.) in small aliquots in orderto avoid free-thaws.

C. Analysis

It was found empirically using HPLC that the extent of modification ofthe alpha-amino group of methionine is substantially greater using thesulfonated form of NHS BODIPY FL compared to the non-sulfonated form ofNHS-BODIPY FL reagent. In addition the misaminoacylated tRNA^(fmet)formed using the sulfonated form was found to exhibit superiorproperties. When used in an optimized S30 E. coli translation systems toincorporate BIDOPY FL into the protein (hemolysin using a plasmidcontaining the HL gene under control of a T7 promoter), the band on anSDS-PAGE gel corresponding to the expressed HL exhibited anapproximately 2 times higher level of fluorescence when detected using aargon laser based fluoroimager compared to a similar system using themisaminoacylated formed using the non-sulfonated form.

D. Coumarin

A similar result to that described above was obtained by comparing thenon-sulfonated and sulfonated NHS derivatives of coumarin, which arealso commercially available and referred to respectively as succinimidyl7-amino-methyl-amino-coumarin acetate (AMCA-NHS; Molecular Probes) andsulfosuccinimidyl 7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS;Pierce Chemicals). In this case, optimized reactions were performedusing these two different reagents based on standard biochemicalprocedures in order to add the coumarin fluorophore to a purifiedtRNA^(fmet) which is aminoacylated with methionine.

To the aminoacylated-tRNA solution described above, 2.5 (1 of 1N NaHCO₃was added (final conc. 50 mM, pH=8.5) followed by 10 (1 of 10 mMsolution of sulfosuccinimidyl 7-amino-4-methylcoumarin-3-acetate(AMCA-sulfo-NHS; Pierce Chemicals) in water. The mixture was incubatedfor 10 min at 0(C and the reaction was quenched by the addition oflysine (final concentration=100 mM). To the resulting solution 0.1volume of 3 M NaOAc, pH=5.0 was added and the modified tRNA wasprecipitated with 3 volumes of ethanol. Precipitate was dissolved in 50microliters of water and purified on Sephadex G-25 gel filtration column(0.5×5 cm) to remove any free fluorescent reagent, if present. Themodified tRNA was stored frozen (−70° C.) in small aliquots in order toavoid free-thaws.

To the above aminoacylated-tRNA solution, 2.5 (1 of 1N NaHCO₃ (finalconc. 50 mM, pH=8.5) and 20 (1 of DMSO was added followed by 10 (1 of 10mM solution of succinimidyl 7-amino-methyl-amino-coumarin acetate(AMCA-NHS; Molecular Probes) in DMSO. The mixture was incubated for 10min at 0° C. and the reaction was quenched by the addition of lysine(final concentration=100 mM). To the resulting solution 0.1 volume of 3M NaOAc, pH=5.0 was added and the modified tRNA was precipitated with 3volumes of ethanol. Precipitate was dissolved in 50 microliter of waterand purified on Sephadex G-25 gel filtration column (0.5×5 cm) to removeany free fluorescent reagent, if present. The modified tRNA was storedfrozen (−70° C.) in small aliquots in order to avoid free-thaws.

In this case, the coumarin-methionine-tRNA^(fmet) formed using thenon-sulfonated form of coumarin-NHS (AMCA-NHS) when used in standard E.coli S30 translation mixtures generated very low levels of detectablefluorescent bands when detected using UV light from a standard UVtransilluminator. In contrast, the sulfonated form (AMCA-sulfo-NHS) whenadded using the same procedures led to easily detectable bands using theUV-transilluminator.

Attempts to incorporate coumarin using an initiator tRNA by modifyingthe α-amino group of methionine have been reported in the literature butfailed. Coumarin attachment to a initiator tRNA subsequently requiredmore extensive and complicated chemical attachment using a chemicalcross linker. This was achieved by first aminoacylating the tRNA withmethionine followed by reaction of aminoacylated tRNA with DTDGmonosuccinimidyl ester (DTDG is Dithiodiglycolic acid). The reactionproduct was then reduced using DTT and subsequently reacted with CPM(3-(4′-Maleimidophenyl)-4-methyl-diethylamino coumarin. (Odom, 0. W,Kudlicki, W and Hardestry, B. 1998. In vitro engineering usingacyl-derivatized tRNA, In Protein synthesis: Methods and Protocols, PP.93-103, Humana press, Totowa, N.J.). Due to the need for specialprocedures designed for each marker, such an approach is not practicalfor general attachment of a wide variety of markers to tRNAs throughpost-chemical aminoacylation procedures.

One likely factor that makes sulfonated NHS reagents used forpostchemical aminoacylation of tRNAs is its solubility in aqueousbuffer. In contrast, non-sulfonated reagents such as the BIDOPY FL NHSreagent require organic buffer such as DMSO for postchemicalmodification. While it is still not clear why use of organic bufferslowers the overall marker incorporation, one possibility is thathydrolysis of the aminoacyl bond formed between the amino acid and tRNAreduces the overall level of modification.

EXAMPLE 15 Imparting Water Solubility

In general, the property of water solubility can be imparted to chemicalreagents in several ways. Some of these are summarized below:

-   -   Introduction of polar functional group into leaving group (such        as sulfonated-NHS).    -   Introduction of the polar functional group into a spacer arm.    -   Introduction of the polar functional group into the reagent        moiety itself.        While the introduction of the —SO₃ ⁻Na⁺ (sulfo-) group is        preferred, other polar ionizable groups (such as DSP) can also        be used where DSP is shown below:

Final water solubility can be engineered into a spacer arm for exampleby using a polyether spacer (e.g. one based on tetraethylene glycol). Ingeneral, any moiety that has a free carboxyl group can be converted intoits sulfo-NHS active ester. This reaction involvesN-hydroxy-sulfosuccinimide (monosodium salt), the marker(^({circle around (M)})) and a coupling agent such as DCC(dicyclohexylcarbodiimide):

In a typical reaction, marker 1, 55 mmol, is dissolved in 10 ml DMF(dimethylformamide) and (2) (5 mmol) is added, followed by (3) (1.1equivalents). The mixture is stirred overnight at room temperature,precipitate filtered off, and the filtrate evaporated under reducedpressure at room temperature. The product is purified if necessary usingcolumn chromatography or is recrystallized.

One preferred embodiment of this invention involves the post-chemicalmodification of tRNAs to form a misaminoacylated tRNA by using markersthat contain a sulfonated NHS reagent. While such reagents are notgenerally available commercially, such reagents can be routinelyproduced out of a variety of useful markers. For example, fluorescein,which has a high fluorescent quantum yield for both UV and visibleexcitation could be prepared in a form which contains a sulfonated NHSester.

EXAMPLE 16 Triple Marker System

In this example, a three marker system is employed to detect nascentproteins, i.e. an N-terminus marker, a C-terminus marker, and anaffinity marker (the latter being an endogenous affinity marker). Theexperiment involves 1) preparation of a tRNA with a marker, so that amarker can be introduced (during translation) at the N-terminus of theprotein; 2) translation of hemolysin with nucleic acid coding for wildtype and mutant hemolysin; and 4) quantitation of the markers.

1. Preparation of Biotin-Methionyl-tRNA^(fmet)

The purified tRNA^(fmet) (Sigma Chemicals, St. Louis, Mo.) was firstaminoacylated with methionine. The typical aminoacylation reactioncontained 1500 picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HClbuffer, pH 7.5, 10 mM MgCl₂, 1 mM methionine, 2 mM ATP, 150 mM NaCl andexcess of aminoacyl tRNA-synthetases (Sigma). The reaction mixture wasincubated for 45 min at 37° C. After incubation, the reaction mixturewas neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 andsubjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5volumes) was added to the aqueous phase and the tRNA pellet obtained wasdissolved in the water (25 μl). The coupling of NHS-biotin to theα-amino group of methionine was carried out in 50 mM sodium bicarbonatebuffer, pH 8.0 by incubating the aminoacylated tRNA^(fmet) (25 μl) withNHS-biotin (final concentration=2 mM) for 10 min at 0° C. and thereaction was quenched by the addition of free lysine (finalconcentration=100 mM). The modified tRNA was precipitated with ethanoland passed through Sephadex G-25 gel filtration column (0.5×5 cm) toremove any free reagent, if present.

2. In Vitro Translation of α-HL DNA

A WT and Amber (at position 135) mutant plasmid DNA was using coding forα-hemolysin (α-HL), a 32 kDa protein bearing amino acid sequenceHis-His-His-His-His-His (His-6) (SEQ ID NO: 5) at its C-terminal. Invitro translation of WT and amber mutant α-HL gene (Amb 135) was carriedout using E. coli T7 circular transcription/translation system (PromegaCorp., Wisconsin, Wis.) in presence of Biotin-methionyl-tRNA^(fmet)(AmberGen, Inc.). The translation reaction of 100 μl contained 30 μl E.coli extract (Promega Corp., Wisconsin, Wis.), 40 μl premix withoutamino acids, 10 μl amino acid mixture (1 mM), 5 μg of plasmid DNA codingfor WT and mutant α-HL, 150 picomoles of biotin-methionyl-tRNA^(fmet)and RNase-free water. The premix (1×) contains 57 mM HEPES, pH 8.2, 36mM ammonium acetate, 210 mM potassium glutamate, 1.7 mM DTT, 4% PEG8000, 1.25 mM ATP, 0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenolpyruvate, 0.6 mM cAMP and 6 mM magnesium acetate. From the translationreaction premix, n-formyl-tetrahydrofolate (fTHF) was omitted. Thetranslation was carried out at 37° C. for 1 hour. The translationreaction mixture incubated without DNA is taken as control. After thetranslation reaction mixture was diluted with equal volume of TBS(Tris-buffered saline, pH 7.5). Each sample was divided into twoaliquots and processed individually as described below.

3. Preparation of Anti-α-HL Antibody Microtiter Plate

Anti-rabbit-IgG coated microtiter plate (Pierce Chemicals, Rockford, II)was washed with Superblock buffer solution (Pierce) and incubated with100 μg/ml of anti-α-HL polyclonal antibody solution (Sigma Chemicals,St. Louis, Mo.) prepared in Superblock buffer on microtiter plate shakerfor 1 hour at room temperature. The plate was then washed (3 times×200μl) with Superblock buffer and stored at 4° C. till further use.

4. Quantitation of N-Terminal (Biotin) Marker

The translation reaction mixture (50 μl) for the control, WT and amberα-HL DNA were incubated in different wells of anti-α-HL microtiter platefor 30 minutes on the shaker at room temperature. After incubation, thewells were washed 5 times (5-10 min each) with 200 μl Superblock bufferand the supernatant were discarded. To these wells, 100 μl of 1:1000diluted streptavidin-horse radish peroxidase (Streptavidin-HRP; 0.25mg/ml; Promega) was added and the plate was incubated at roomtemperature for 20 min under shaking conditions. After the incubation,excess streptavidin-HRP was removed by extensive washing with Superblockbuffer (5 times×5 min each). Finally, 200 μl of substrate for HRP (OPDin HRP buffer; Pierce) was added and the HRP activity was determinedusing spectrophotometer by measuring absorbance at 441 nm.

5. Quantitation of C-Terminal (His-6-taq) Marker

Translation reaction mixture (50 μl) from example 2 for control, WT andAmber α-HL DNA were incubated in different wells of anti-α-HL microtiterplate for 30 min on the shaker at room temperature. After incubation,the wells were washed 5 times (5-10 min each) with 200 μl Superblockbuffer and the supernatant were discarded. To these wells, 100 μl of1:1000 diluted anti-His-6 antibody (ClonTech, Palo Alto, Calif.) wasadded to the well and incubated at room temperature for 20 min undershaking conditions. After the incubation, excess antibodies were removedwith extensive washing with Superblock buffer (5 times×5 min each).Subsequently, the wells were incubated with secondary antibody(anti-mouse IgG-HRP, Roche-BM, Indianapolis, Ind.) for 20 min at roomtemperature. After washing excess 2^(nd) antibodies, HRP activity wasdetermined as described above.

6. Gel-Free Quantitation of N- and C-Terminal Markers

The results of the above-described quantitation are shown in FIG. 23A(quantitation of N-terminal, Biotin marker) and FIG. 23B (quantitationof C-terminal, His-6 marker). In case of in vitrotranscription/translation of WT α-HL DNA in presence ofbiotin-methionyl-tRNA, the protein synthesized will have translatedHis-6 tag at the C-terminal of the protein and some of the α-HLmolecules will also carry biotin at their N-terminus which has beenincorporated using biotinylated-methionine-tRNA. When the totaltranslation reaction mixture containing α-HL was incubated on anti-α-HLantibody plate, selectively all the α-HL will bind to the plate viainteraction of the antibody with the endogenous affinity marker. Theunbound proteins can be washed away and the N- and C-terminal of thebound protein can be quantitated using Streptavidin-HRP and anti-His-6antibodies, respectively. In case of WT α-HL, the protein will carryboth the N-terminal (biotin) and C-terminal (His-6) tags and hence itwill produce HRP signal in both the cases where streptavidin-HRP andsecondary antibody-HRP conjugates against His-6 antibody used (HL, FIG.23A). On the other hand, in case of amber mutant α-HL, only N-terminalfragment of α-HL (first 134 amino acids) will be produced and will haveonly N-terminal marker, biotin, but will not have His-6 marker due toamber mutation at codon number 135. As a result of this mutation, theprotein produced using amber α-HL DNA will bind to the antibody platebut will only produce a signal in the case of strepavidin-HRP (HL-AMB,FIG. 23A) and not for anti-H is ×6 antibodies (HL-AMB, FIG. 23B).

EXAMPLE 17 Electrophoretic Mobility Shift Assay

To demonstrate the changes in the electrophoretic mobility offluorescently labeled nascent protein on the SDS-gels either due toproteolysis or oligomerization in presence of membranes, we have useplasmid DNA of α-hemolysin (α-HL) which codes 32 kDa protein bearing asequence His-His-His-His-His-His (His-6) (SEQ ID NO:5) at itsC-terminal. In vitro translation of α-HL gene was carried out using E.coli T7 circular transcription/translation system (Promega Corp.,Wisconsin-Madison, Wis.) in presence of BODIPY-FL-methionyl-tRNA^(fmet)(AmberGen, Inc.) This experiment involved 1) preparation of thetRNA-marker for introduction of the N-terminus marker duringtranslation, 2) translation, 3) purification, 4) protease treatment or5) oligomerization.

1. Preparation of BODIPY-FL-Methionyl-tRNA:

BODIPY-FL-methionyl-tRNA was prepared by first aminoacylating puretRNA^(fmet) (Sigma Chemicals, St. Louis, Mo.) using methionine andsubsequently modifying α-amino group of methionine using BODIPY-FL-SSE(4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene propionic acid,sulfosuccinimidyl ester; Molecular Probes, Eugene, Oreg.). The typicalaminoacylation reaction (100 μl) contained 1500 picomoles (−1.0 OD₂₆₀)of tRNA, 20 mM imidazole-HCl buffer, pH 7.5, 10 mM MgCl₂, 1 mMmethionine, 2 mM ATP, 150 mM NaCl and excess of aminoacyltRNA-synthetases (Sigma). The reaction mixture was incubated for 45 minat 37° C. After incubation, the reaction mixture was neutralized byadding 0.1 volume of 3 M sodium acetate, pH 5.0 and subjected tochloroform:acid phenol extraction (1:1). Ethanol (2.5 volumes) was addedto the aqueous phase and the tRNA pellet obtained was dissolved in thewater (25 μl). The coupling of BODIPY-FL-SSE to the α-amino group ofmethionine was carried out in 50 μl reaction volume using 50 mM sodiumbicarbonate buffer, pH 8.0 by incubating 25 μl aminoacylated tRNA^(fmet)(1.5 nanomoles) with 10 μl of BODIPY-FL-SSE (10 mM) for 10 min at 0° C.and the reaction was quenched by the addition of free lysine (finalconcentration=100 mM). The modified tRNA was precipitated with ethanol,and the pellet was dissolved in RNase-free water and passed throughSephadex G-25 gel filtration column (0.5×5 cm) to remove any freefluorescent reagent, if present.

2. In Vitro Translation of α-Hemolysin DNA

The translation reaction of 100 μL contained 30 μl E. coli extract(Promega Corp., Wisconsin, Wis.), 40 μl premix without amino acids, 10μl amino acid mixture (1 mM), 5 μg of plasmid DNA coding for α-HL, 150picomoles of BODIPY-FL-methionyl-tRNA^(fmet) and RNase free water. Thepremix (1×) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate, 210 mMpotassium glutamate, 1.7 mM DTT, 4% PEG 8000. 1.25 mM ATP, 0.8 mM GTP,0.8 mM UTP, 60 mM phosphoenol pyruvate, 0.6 mM cAMP and 6 mM magnesiumacetate. From the translation reaction premix, n-formyl-tetrahydrofolate(fTHF) was omitted. The translation was carried out at 37° C. for 1hour. The translation reaction mixture incubated without DNA is taken ascontrol.

3. Purification of Hls-6-α-α-HL

Fifty microliters of the translation reaction mixture (from above) wassubjected to Talon-Sepharose (ClonTech, Palo Alto, Calif.)chromatography for the purification of Hls-6-α-HL. This was carried outby loading the crude extract onto the Talon-Sepharose column which waspre-equilibrated with 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl andwashing the column to remove unbound proteins. The bound protein wasthen eluted by adding 100 mM imidazole in the above buffer. The elutedα-HL was dialyzed against 50 mM Tris-HCl buffer, pH 7.5.

4. EMSA for Protease Detection

The purified fluorescently labeled α-HL (−5 μg) (example 3) wasincubated with 0.0.5 μg of pure trypsin (Sigma Chemicals, St. Louis,Mo.) in 50 nM acetate buffer, pH 5.0 (100:1; protein:protease ratio) for5 min at 37° C. The proteolysis reaction was arrested by the addition of1×SDS-gel loading buffer and boiling the samples for 5 min. The SDS-PAGEwas carried out as described by Laemmli (Laemmli, U. K. 1970, Nature227, 680-685) using 4-20% gradient gel (ready-gel, Bio-Rad, Richmond,Calif.). After the gel electropboresis, the gel was visualized usingFluorlmager F595 (Molecular Dynamics, Sunnyvale, Calif.).

Trypsin was used under very limited conditions (single-hit kinetics) toobtain very defined cleavage of α-HL (50 mM acetate buffer, pH 5.0,100:1: protein:protease ratio, 5 min at 37° C.). Under these conditions,the glycine rich loop in α-HL is most susceptible to cleavage and as aresult proteolytic fragment of 17 kDa was observed (Vecesey-Semjen, B.,Knapp, S., Mollby, R., Goot, F. G. 1999, Biochemistry, 38 4296-4302).When the fluorescently labeled α-HL was subjected to very mild trypsintreatment, it resulted a cleavage of α-HL yielding the N-terminalfragment of approximately 17-18 kDa mass as evidence by change in themobility of fluorescent band on SDS-PAGE (FIG. 24: Lane 1 showsuntreated protein and Lane 2 shows protease treated protein). Thisresult indicates that such assay could be used to screen for proteasesor any other enzymatic activities like kinase, transferase etc. thatcould potentially result in the electrophoretic mobility shift of thenascent protein. Though we have used pure nascent protein for thisparticular assay, there is no reason why one can not use a nascentprotein without any purification (total translation reaction mixture).

5. EMSA for Oligomerization of Nascent Protein on Membranes

The total translation reaction mixture (10 μl) (see above) was incubatedin absence and in presence of rabbit red blood cells (rRBCs, CharlesRiver Farm, Conn.) for 30 min. at 0° C. After the incubation, rRBCs werewashed free of excess unbound α-HL and the rRBCs were incubated in Trisbuffer saline (TBS) containing 1 mg/ml BSA (TBSA) at 37° C. for 20 minduring which lysis of rRBCs occurred. The rRBC membranes were isolatedafter centrifugation, dissolved in 1×SDS-gel loading buffer andsubjected to SDS-PAGE (4-20% gradient gel) without heating the sample.After the gel electrophoresis, the gel was visualized using FluorImagerF595.

α-HL is expressed a soluble monomeric protein and in presence of variousmembranes it can oligomerize to form heptameric pore (Walker, B.,Krishnasastry, M., Zorn, L., Kasianowicz, J. and Bayley, H., 1992, J.Biol. Chem. 267, 10902-10909). In addition, some intermediate forms ofthe oligomers were also observed. In this experiment, in order to seethe applicability of EMSA to detect the shift in mobility of α-HL due tooligomerization in presence lipid membranes, the total translationreaction mixture (with out any purification) was used. When the totaltranslation extract containing nascent α-HL was incubated with rRBCs, itresulted in the oligomerization of α-HL on the rRBC membranes yielding adistinct fluorescent bands corresponding to various molecular massesthat were SDS-resistant (FIG. 25: Lane 1 shows untreated protein onlyand Lane 2 shows protein treated with rRBCs).

This result demonstrates that such assay could be used to studyproteins, interact with variety of natural and artificial membranes andas a result the mobility of the protein in shifted.

EXAMPLE 18 Incorporation Using Lysyl-tRNA^(lys)

This example describes the incorporation of fluorescent labels intonascent protein using lysyl-tRNA^(lys). More specifically, a variety offluorescent molecules were incorporated into 1) hemolysin duringtranslation in an E coli translation system, and 2) luciferase duringtranslation in a wheat germ system, using lysyl tRNA^(lys). Theexperiment involved 1) preparation of the tRNA-marker compounds, 2)translation, and 3) detection on gels.

1. Preparation of Fluorescent Labeled Misaminoacylated tRNA^(lys)

The purified tRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was firstamino-acylated with lysine. The typical aminoacylation reaction (100 μl)contained 1500 picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HClbuffer, pH 7.5, 10 mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl andexcess of aminoacyl tRNA-synthetases (Sigma). The reaction mixture wasincubated for 45 min at 37° C. After incubation, the reaction mixturewas neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 andsubjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5volumes) was added to the aqueous phase and the tRNA pellet obtained wasdissolved in the water (25 μl). The coupling of NHS-derivatives ofvarious fluorescent molecules (see Table 2) to the α-amino group oflysine was carried out in 50 mM CAPS buffer, pH 10.5 by incubating theaminoacylated tRNA^(lys) (25 μl) with fluorescent reagent (finalconcentration=2 mM) for 10 min at 0° C. and the reaction was quenched bythe addition of free lysine (final concentration=100 mM). The modifiedtRNA was precipitated with ethanol, dissolved in 50 μl of RNase-freewater and passed through Sephadex G-25 gel filtration column (0.5×5 cm)to remove any free fluorescent reagent, if present. The modified tRNAwas stored frozen (−70° C.) in small aliquots in order to avoidfree-thaws. The modification extent of the aminoacylated-tRNA wasassessed by acid-urea gel electrophoresis (Varshney, U., Lee, C. P. &RajBhandary, U. L., 1991 J. Biol. Chem. 266, 24712-24718).

2. Cell Free Synthesis of Proteins in Prokaryotic (E. coli) TranslationExtracts

The typical translation reaction mixture (10 μl) contained 3 μl of E.coli extract (Promega Corp., Wisconsin-Madison, Wis.), 4 μl of premix, 1μl of amino acid mix (1 mM), 30 picomoles of fluorescent-lysyl-tRNA and0.5 μg of a hemolysin (αHL) plasmid DNA. The premix (1×) contains 57 mMHEPES, pH 8.2, 36 mM ammonium acetate, 210 mM potassium glutamate, 1.7mM DTT, 4% PEG 8000, 1.25 mM ATP, 0.8 mM GTP, 0.8 mM CTP, 60 mMphosphoenolpyruvate, 0.6 mM cAMP and 6 mM magnesium acetate. Thetranslation reaction was allowed to proceed for 45 min at 37° C. ForSDS-PAGE, 4-10 μl aliquot of the reaction mix was precipitated with5-volume acetone and the precipitated proteins were collected bycentrifugation. The pellet was dissolved in 1×loading buffer andsubjected to SDS-PAGE after boiling for 5 min. SDS-PAGE was carried outaccording to Laemmmli (Lammli, U.K. 1970, Nature, 227, 680-685).

3. Cell-Free Synthesis in Eukaryotic (TnT Wheat Germ) TranslationExtracts.

The typical translation reaction mixture (10 μl) contained 5 μl of TnTwheat germ extract (Promega Corp., Wisconsin-Madison, Wis.), 0.4 μl ofTnT reaction buffer, 1 μl of amino acid mix (1 mM), 0.2 μl of T7 RNApolymerase, 30 picomoles of fluorescent-lysyl-tRNA and 0.5 μg ofluciferase RNA (Promega) and RNase-free water. The translation reactionwas allowed to proceed for 45 min at 30° C. and reaction mixture wascentrifuged for 5 min to remove insoluble material. The clarifiedextract was then precipitated with 5-volume acetone and the precipitatedproteins were collected by centrifugation. The pellet was dissolved in1× loading buffer and subjected to SDS-PAGE after boiling for 5 min.SDS-PAGE was carried out according to Laemmli (Lammli, U.K. 1970),Nature, 227, 680-685).

4. Detection of Nascent Protein

The gel containing nascent proteins was scanned using FluorImager F595(Molecular Dynamics, Sunnyvale, Calif. using Argon laser (488 nm) asexcitation source, in addition, the nascent proteins in polyacrylamidegels were also detected using an UV-transilluminator and the photographswere carried out using Polaroid camera fitted with Tiffen green filter(Polaroid, Cambridge, Mass.). FIGS. 26A and 26B show the results of invitro translation of α-HL produced in presence of various fluorescenttRNA^(lys). It is clear from the results one can incorporate a varietyof fluorescent molecules into nascent protein using misaminoacylatedtRNA (fluorophore-modified lysyl-tRNA^(lys)) including dyes like NBD,fluorescein derivatives etc. (Lane 1: No DNA control; lane 2:BODIPY-FL-SSE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenepropionic acid, sulfosuccinimidyl ester); lane 3: BODIPY-FL-SE(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene propionic acid,succinimidyl ester); lane 4: NBD-X-SE (Succinimidyl6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoate); lane 5;BODIPY-TMR-SE((6-994,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoic acid, succinimidyl ester); lane 6: BODIPY-R6G-SE((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid,succinimidyl ester); lane 7; FAM-SE (5-(6-)-carboxyfluorescein,succinimidyl ester); lane 8:SFX-SE (6-fluorescein-5-(and6-)carboxyamido)hexonoic acid, succinimidyl ester); lane 9: PyMPO-SE(1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridininiumbromide (PyMPO)) and lane 10: TAMRA-SE(6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid,succinimidyl ester).

EXAMPLE 19 NHS-Derivatives of Coumarin

Given the above noted results for the two BODIPY molecules (i.e.BODIPY-FL-SSE and BODIPY-FL-SE), attempts were made to derivatize othermarkers to make them suitable for incorporation. The present exampleinvolves 1) the preparation of the labeled tRNA, 2) translation, and 3)detection of the nascent protein containing the label (or marker).

1. Preparation of Fluorescent Labeled Misaminoacylated tRNA^(fmet)

The purified tRNA^(fmet) (Sigma Chemicals, St. Louis, Mo.) was firstaminoacylated with methionine. The typical aminoacylation reaction (100μl) contained 1500 picomoles (approximately 1.0 OD₂₆₀) of tRNA, 20 mMimidazole-HCl buffer, pH 7.5, 10 MM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mMNaCl and excess of aminoacyl tRNA-synthetases (Sigma). The reactionmixture was incubated for 45 min at 37° C. After incubation, thereaction mixture was neutralized by adding 0.1 volume of 3 M sodiumacetate, pH 5.0 and subjected to chloroform:acid phenol extraction(1:1). Ethanol (2.5 volumes) was added to the aqueous phase and the tRNApellet obtained was dissolved in the water (25 μl). The coupling ofNHS-derivatives of coumarin [namely sulfosuccinimidyl7-amino-4-methylcoumarin-3-acetate [1] (AMCA-sulfo-NHS; PierceChemicals), Alexa 350-N-hydroxy-succinimide ester (Molecular Probes) andsuccinimidyl 7-amethyl-amino-coumarin acetate (AMCA-NHS: MolecularProbes)] to the α-amino group of methionine was carried out in 50 mMsodium bicarbonate buffer, pH 8.5 by incubating the aminoacylatedtRNA^(fmet) (25 μl) with fluorescent reagent (final concentration=2 mM)for 10 min at 0° C. and the reaction was quenched by the addition offree lysine (final concentration=100 mM). In case of AMCA-NHS, reagentwas dissolved in DMSO and the coupling reaction was carried out inpresence of 40% DMSO. The modified tRNA was precipitated with ethanol,dissolved in 50 μl of RNase-free water and passed through Sephadex G-25gel filtration column (0.5×5 cm) to remove any free fluorescent reagent,if present. The modified tRNA was stored frozen (−70° C.) in smallaliquots in order to avoid free-thaws. The modification extent of theaminoacylated-tRNA was assessed by acid-urea gel electrophoresis(Varshney, U., Lee, C. P. & RajBhandary, U. L., 1991, J. Biol. Chem.266, 24712-24718).

2. Cell Free Synthesis of Proteins in Prokaryotic (E. coli) TranslationExtracts:

The typical translation reaction mixture (10 μl) contained 3 μl of E.coli S-30 extract (Promega Corp., Wisconsin-Madison, Wis.), 4 μl ofpremix, 1 μl of amino acid mix (1 mM), 30 picomoles offluorescent-methionyl-tRNA and 0.5 μg of α-hemolysin (α-HL) plasmid DNA.The premix (1×) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate,210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP, 0.8 mMGTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6 mM cAMP and6 mM magnesium acetate. The translation reaction was allowed to proceedfor 45 min at 37° C. For SDS-PAGE, 4-10 μl aliquot of the reaction mixwas precipitated with 5-volume acetone and the precipitated proteinswere collected by centrifugation. The pellet was dissolved in IX loadingbuffer and subjected to SDS-PAGE after boiling for 5 min. SDS-PAGE wascarried out according to Laemmli (Laemmli, U. K. 1970, Nature, 277,680-685).

3. Detection of Nascent Protein

The gel containing nascent proteins was visualized using anUV-transilluminator equipped with long wavelength UV bulb (>300 nm) andthe photographs were carried out using Polaroid camera fitted withTiffen green filter (Polaroid, Cambridge, Mass.). FIG. 27 indicates thatthe result in vitro translation of α-HL produced in presence of variousfluorescent tRNAs (Lane 1 shows the results for the no DNA control; Lane2 shows the results with Met-tRNA^(fmet) modified with AMCA-NHS; Lane 3shows the results with Met-tRNA^(fmet) modified with AMCA-sulfo-NHS; andLane 4 shows the results with Met-tRNA^(fmet) modified with Alexa-NHS).Clearly, one can incorporate the coumarin derivative molecules intonascent protein using misaminoacylated tRNA which are modified with thewater soluble NHS-esters of fluorescent molecules (lane 3). Moreover, adye with negative charge (Alexa, lane 4) seems to not incorporate aswell as its neutral counterpart (AMCA; lane 3).

From the above results and general teachings of the presentspecification, one skilled in the art can select other markers andrender them (e.g. chemically render them) suitable for incorporation inaccordance with the methods of the present invention.

EXAMPLE 20 Capillary Electrophoresis

The example describes the use of capillary electrophoresis (CE) fordetection of in vitro synthesized fluorescent proteins by mobilityshift. The example describes 1) the preparation of the tRNA comprising aBODIPY marker, 2) in vitro translation, 3) purification, 4) proteasedigestion and 5) detection by mobility shift assay.

1. Preparation of BODIPY-FL-Methionyl-tRNA^(fmet)

The tRNA^(fmet) was amino acylated with the methionine. In typicalreaction, 1500 picomoles (˜1.0 OD₂₆₀) of tRNA was incubated for 45 minat 37° C. in aminoacylation mix using an excess of aminoacyltRNA-synthetases. The aminoacylation mix comprised 20 mM imidazole-HClbuffer, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 2 mM ATP and 1600 units ofaminoacyl tRNA-synthetase. The extent of aminoacylation was determinedby acid-urea gel as well as by using ³⁵ S-methionine. After incubation,the mixture was neutralized by adding 0.1 volume of 3 M sodium acetate,pH 5.0 and subjected to chloroform:acid phenol (pH 5.0) extraction(1:1). Ethanol (2.5 volumes) was added to the aqueous phase and the tRNApellet obtained was dissolved in water (37.5 ul) and used formodification. To the above aminoacylated-tRNA solution, 2.5 ul of 1NNaHCO₃ was added (final conc. 50 mM, pH=8.5) followed by 10 ul of 10 mMsolution of BODIPY-FL-SSE (Molecular Probes) in water. The mixture wasincubated for 10 min at 0° C. and the reaction was quenched by theaddition of lysine (final concentration=100 mM). To the resultingsolution 0.1 volume of 3 M NaOAc, pH=5.0 was added and the modified tRNAwas precipitated with 3 volumes of ethanol. Precipitate was dissolved in50 μl of water and purified on Sephadex-G-25 gel filtration column(0.5×5 cm) to remove any free fluorescent reagent, if present. Themodified tRNA was stored frozen (−70° C.) in small aliquots in order toavoid free-thaws.

2. In Vitro Translation of α-Hemolysin DNA

The translation reaction of 500 ul contained 150 ul E. coli extract(Promega Corp., Wisconsin, Wis.), 200 ul premix without amino acids, 50ul amino acid mixture (1 mM), 25 ug of plasmid DNA coding for α-HL, 1000picomoles of BODIPY-FL-methionyl-tRNA^(fmet) and RNase free water. Thepremix (1×) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate, 210 mMpotassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP, 0.8 mM GTP,0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6 mM cAMP and 16mM magnesium acetate. From the translation reaction premix,n-formyl-tetrahydrofolate (fTHF) was omitted. The translation wascarried out at 37° C. for 1 hour. The translation reaction mixtureincubated without DNA is taken as control.

3. Purification of His-6-α-HL

Five hundred microliters of the translation reaction mixture (see step 2above) was subjected to Talon-Sepharose (ClonTech, Polo Alto, Calif.)chromatography for the purification of His-6-α-HL. This was carried outby loading the crude extract onto the Talon-Sepharose column which waspre-equilibrated with 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl andwashing the column to remove unbound proteins. The bound protein wasthen eluted by adding 100 mM imidazole in the above buffer. The elutedα-HL was dialyzed against 50 mM Tris-HCl buffer, pH 7.5. Thefluorescence of purified and dialyzed α-HL was checked on MolecularDynamics FluorImager F595.

4. Protease Digestion

The purified fluorescently labeled α-HL (˜5 ug) (see step 3 above) wasincubated with 0.1 ug of pure trypsin (Sigma Chemicals, St. Louis, Mo.)in 50 mM Tris-HCl buffer, pH 7.5 (50:1; protein:protease ratio) for 10min at room temperature. The proteolysis reaction was arrested by theaddition of 1×CE-SDS-gel loading buffer and boiling the samples for 10min.

5. Mobility Shift Assay

The SDS-capillary gel electrophoresis (SDS-CGE) was performed on aBio-Rad BioFocus 3000CE system. The capillary was fused-silica with a 75um ID, 24 cm total length and 19.5 cm to the detector. Fifty microlitersof fluorescently labeled protein sample (α-HL) was mixed with 50 ul ofSDS-CGE-sample loading buffer and incubated at 95° C. for 10 min. Thecapillary was rinsed with 0.1 M NaOH, 0.1 M HCl and SDS-run buffer for60, 60 and 120 sec respectively, prior to each injection. Sample wereinjected using electrophoretic injection (20 sec at 10 kV). Separationwas performed at 15 kV (constant voltage) for 25 min. Capillary andsample was maintained at 20° C. The detection of sample was carried outusing 488 nm Argon laser and 520 nm emission filter.

The results of SDS-CGE are shown in the FIG. 28. As seen in the Figure,fluorescently label α-HL migrates as a major species eluting around 24min under the electrophoresis conditions (Top panel). In addition, theelectrophoregram also show the presence of minor impurities present inthe sample, which are eluting around 17 and 20.5 min. When thefluorescently labeled-α-HL sample was treated with trypsin and analyzedusing SDS-CGE, it showed peaks eluting earlier (13, 14 and 15 min) andmajor peak at 21 min (Bottom panel). This result indicated that the α-HLwas proteolysed by the trypsin and various proteolytic fragments haveN-terminal fluorescently labeled are seen in the electrophoregram.

EXAMPLE 21 Incorporation of Three Markers into Hemolysin

This is an example wherein a protein is generated in vitro underconditions where N- and C-terminal markers are incorporated along with amarker incorporated using a misaminoacylated tRNA. The Exampleinvolves 1) PCR with primers harboring N-terminal and C-terminaldetectable markers, 2) preparation of the tRNA, 3) in vitro translation,4) detection of nascent protein.

1. PCR of α-Hemolysin DNA

Plasmid DNA for α-hemolysin, pT7-WT-H6-αHL, was amplified by PCR usingfollowing primers. The forward primer (HL-5) was:5′-GAATTC-TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGGAACAAAAATTAATCTCGGAAGAGGATTTGGCAGATTCTGATATTAATATTAAAACC-3′ (SEQ ID NO:11)and the reverse primer (HL-3) was: 5′-AGCTTCATTA-ATGATGGTGATGG-TGGTGAC3′ (SEQ ID NO:12). The underlined sequence in forward primer is T7promoter, the region in bold corresponds to ribosome binding site(Shine-Dalgarno's sequence), the bold and underlined sequences involvethe C-myc epitope and nucleotides shown in italics are the complimentaryregion of α-hemolysin sequence. In the reverse primer, the underlinedsequence corresponds to that of His×6 epitope. The PCR reaction mixtureof 100 ul contained 100 ng template DNA, 0.5 uM each primer, 1 mM MgCl₂,50 ul of PCR master mix (Qiagen, CA.) and nuclease free water (SigmaChemicals, St. Louis, Mo.) water. The PCR was carried out using HybaidOmni-E thermocyler (Hybaid, Franklin, Mass.) fitted with hot-lid usingfollowing conditions: 95° C. for 2 min, followed by 35 cycles consistedof 95° C. for 1 min, 61° C. for 1 min and 72° C. for 2 min and the finalextension at 72° C. for 7 min. The PCR product was then purified usingQiagen PCR clean-up kit (Qiagen, CA.). The purified PCR DNA was used inthe translation reaction.

2. Preparation of BODIPY-FL-lysyl-tRNA^(lys)

The purified tRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was firstaminoacylated with lysine. The typical aminoacylation reaction contained1500 picomoles (˜1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HCl buffer, pH 7.5,10 mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl and excess of aminoacyltRNA-synthetases (Sigma Chemicals, St. Louis, Mo.). The reaction mixturewas incubated for 45 min at 37° C. After incubation, the reactionmixture was neutralized by adding 0.1 volume of 3 M sodium acetate, pH5.0 and subjected to chloroform:acid phenol extraction (1:1). Ethanol(2.5 volumes) was added to the aqueous phase and the tRNA pelletobtained was dissolved in water (35 ul). To this solution 5 ul of 0.5 MCAPS buffer, pH 10.5 was added (50 mM final conc.) followed by 10 ul of10 mM solution of BODIPY-FL-SSE. The mixture was incubated for 10 min at0° C. and the reaction was quenched by the addition of lysine (finalconcentration 100 mM). To the resulting solution 0.1 volume of 3 MNaOAc, pH=5.0 was added and the modified tRNA was precipitated with 3volumes of ethanol. Precipitate was dissolved in 50 ul of water andpurified on Sephadex G-25 gel filtration column (0.5×5 cm) to remove anyfree fluorescent reagent, if present. The modified tRNA was storedfrozen (−70° C.) in small aliquots in order to avoid free-thaws. Themodification extent of the aminoacylated-tRNA was assessed by acid-ureagel electrophoresis. Varshney et al., J. Biol. Chem. 266:24712-24718(1991).

3. Cell-Free Synthesis of Proteins in Eukaryotic (Wheat Germ)Translation Extracts.

The typical translation reaction mixture (20 ul) contained 10 ul of TnTwheat germ extract (Promega Corp., Wisconsin-Madison, Wis.), 0.8 ul ofTnT reaction buffer, 2 ul of amino acid mix (1 mM), 0.4 ul of T7 RNApolymerase, 30 picomoles of BODIPY-FL-lysyl-tRNA^(lys), 1-2 ug plasmidor PCR DNA (Example 1) and RNase-free water. The translation reactionwas allowed to proceed for 60 min at 30° C. and reaction mixture wascentrifuged for 5 min to remove insoluble material. The clarifiedextract was then precipitated with 5-volumes of acetone and theprecipitated proteins were collected by centrifugation. The pellet wasdissolved in 1× loading buffer and subjected to SDS-PAGE after boilingfor 5 min. SDS-PAGE was carried out according to Laemmli, Nature,227:680-685.

4. Detection of Nascent Protein

After the electrophoresis, gel was scanned using FluorImager 595(Molecular Dymanics, Sunnyvale, Calif.) equipped with argon laser asexcitation source. For visualization of BODIPY-FL labeled nascentprotein, we have used 488 nm as the excitation source as it is theclosest to its excitation maximum and for emission, we have used530+/−30 filter. The gel was scanned using PMT voltage 1000 volts andeither 100 or 200 micron pixel size.

The results are shown in FIG. 29. It can be seen from the Figure thatone can in vitro produce the protein from the PCR DNA containing desiredmarker(s) present. In the present case, the protein (α-hemolysin) has aC-myc epitope at N-terminal and His×6 epitope at C-terminal. Inaddition, BODIPY-FL, a fluorescent reporter molecule is incorporatedinto the protein. Lane 1: α-Hemolysin plasmid DNA control; lane 2: noDNA control; lane 3: PCR α-hemolysin DNA and lane 4: hemolysin amber 135DNA. The top (T) and bottom (B) bands in all the lane are from thenon-specific binding of fluorescent tRNA to some proteins in wheat germextract and free fluorescent-tRNA present in the translation reaction,respectively.

EXAMPLE 22 Primer Design

It is not intended that the present invention be limited to particularprimers. A variety of primers are contemplated for use in the presentinvention to ultimately incorporate markers in the nascent protein (asexplained above). The Example involves 1) PCR with primers harboringmarkers, 2) in vitro translation, and 3) detection of nascent protein.

For PCR the following primers were used:

forward primer: (SEQ ID NO:13) 5′ GGATCC TAATACGACTCACTATAG

CCACCATG

GTTTCTCCATACAGGTCACGGGGA- 3′. Reverse primer: (SEQ ID NO:14)5′-TTATTAATGATGGTGATGGTGGTG- TTCTGTAGGAATGGTATCTCG TTTTTC -3′sequence in the forward primer is T7 promoter, the bold and underlinedsequences involve the C-myc epitope and nucleotides shown in italics arethe complimentary region of α-hemolysin sequence. In the reverse primer,the bold sequence corresponds to that of His-6 epitope and theunderlined sequence corresponds to the complimentary region of theα-hemolysin sequence. A PCR reaction mixture of 100 ul can be usedcontaining 100 ng template DNA, 0.5 uM each primer, 1 mM MgCl₂, 50 ul ofPCR master mix (Qiagen, CA.) and nuclease free water (Sigma Chemicals,St. Louis, Mo.) water. The PCR was carried out using Hybaid Omni-Ethermocyler (Hybaid, Franklin, Mass.) fitted with hot-lid usingfollowing conditions: 95° C. for 2 min, followed by 35 cycles consistedof 95° C. for 1 min, 61° C. for 1 min and 72° C. for 2 min and the finalextension at 72° C. for 7 min. The PCR product can then be purifiedusing Qiagen PCR clean-up kit (Qiagen, CA.). The purified PCR DNA canthen be used in a variety of translation reactions. Detection can bedone as described above.

Overall, the present invention contemplates a variety of primer designsbased on the particular epitopes desired (see Table 4 for a list ofillustrative epitopes). In general, the epitopes can be inserted as theN-terminus or C-terminus. In addition, they can be used to introduce anaffinity region (i.e. a region which will bind to antibody or otherligand) into the protein.

EXAMPLE 23 Antibody Detection of Primer-Encoded Epitopes

This is an example wherein a protein is generated in vitro underconditions where affinity regions are incorporated in a protein andthereafter detected. The Example involves 1) PCR with primers containingsequences that encode epitopes, 2) preparation of the tRNA, 3) in vitrotranslation, 4) detection of nascent protein.

1. PCR with Primer-Encoded Epitopes

The total RNA from the human colon (Clontech, Palo Alto, Calif.) wassubjected to one-step RT-PCR reaction using ClonTech RT-PCR Kit. Theforward Primer, PTT-T7-P53, was5′-GGATCCTAATACGACTCACTATAGGGAGACCACCA-TGGGACACCACCATCACCATCACGGAGATTACAAAGATGACGATGACAAA-GAGGAGCCGCAGTCAGATCCTAGCGTCGA-3′(SEQ ID NO:15) and the reverse primer, Myc-P53-3′, was5′-ATTATTACAAATCCTCTTCCGAGATTAATTTTTGTTCGTCTGAGTCAGGCCCTTCTGTCTTGAACATG-3′(SEQ ID NO:16). The underlined sequence in forward primer is T7promoter, the nucleotides shown in italics corresponds to that of His-6tag while the sequence in bold codes for FLAG-epitope and the rest ofprimer is the complementary region for P53 DNA. In the reverse primer,the underlined sequence corresponds to that of c-Myc epitope.

The RT-PCR/PCR reaction mixture of 50 μl contained 1 μg total humancolon RNA, 0.5 μM each primer, 43.5 μl of RT-PCR master mix (ClonTech)and nuclease free water (Sigma Chemicals, St. Louis, Mo.) water. TheRT-PCR/PCR was carried out in PTC-150 thermocyler (MJ Research, Waltham,Mass.) using following conditions: 50° C. for 1 hour, 95° C. for 5 minfollowed by 40 cycles consisted of 95° C. for 45 sec, 60° C. for 1 minand 70° C. for 2 min and the final extension at 70° C. for 7 min. ThePCR product was analyzed on 1% agarose gel and the PCR amplified DNA wasused in the translation reaction without any further purification. Theartificial C-terminal truncated mutant of P53 was prepared using theidentical procedure described above except the reverse primer,3′-P53-Mut, was 5′-CTCATTCAGCTCTCGGAACATC-TCGAAGCG-3′ (SEQ ID NO: 17).

2. tRNA Labelling

Purified tRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was firstamino-acylated with lysine. The typical aminoacylation reaction (100 μl)contained 1500 picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HClbuffer, pH 7.5, 10 mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl andexcess of aminoacyl tRNA-synthetases (Sigma). The reaction mixture wasincubated for 45 min at 37° C. After incubation, the reaction mixturewas neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 andsubjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5volumes) was added to the aqueous phase and the tRNA pellet obtained wasdissolved in the water (35 μl). To this solution 5 ul of 0.5M CAPSbuffer, pH 10.5 was added (final concentration of 50 mM) followed by 10ul of 10 mM solution of BODIPY-FL-SSE. The mixture was incubated for 10minutes at 0° C. and the reaction was quenched by the addition of freelysine (final concentration=100 mM). To the resulting solution 0.1volumes of 3 M NAOAc (pH=5.0) was added and the modified tRNA wasprecipitated with 3 volumes of ethanol. Precipitate was dissolved in 50μl of RNase-free water and passed through Sephadex G-25 gel filtrationcolumn (0.5×5 cm) to remove any free fluorescent reagent, if present.The modified tRNA was stored frozen (−70° C.) in small aliquots in orderto avoid freeze-thaws. The modification extent of the aminoacylated-tRNAwas assessed by acid-urea gel electrophoresis [Varshney, U., Lee, C. P.& RajBhandary, U. L., J. Biol. Chem. 266, 24712-24718 (1991)] or by HPLC[Anal. Biochem. 279:218-225 (2000)].

3. Translation

Translation of P53 DNA (see step 1, above) was carried out in rabbitreticulocyte translation extract in presence of fluorescent-tRNA (step2, above).

4. Detection

Once the translation was over, an aliquot (5 μl) was subjected toSDS-PAGE and the nascent proteins were visualized using FluorImager SI(Molecular Dynamics, Sunnyvale, Calif.). After visualization, the gelwas soaked in the transfer buffer (12 mM Tris, 100 mM glycine and 0.01%SDS, pH 8.5) for 10 min. Proteins from the gels were then transferred toPVDF membrane by standard western blotting protocol using Bio-Radsubmersion transfer unit for 1 hr. After the transfer, then membrane wasreversibly stained using Ferrozine/ferrous total protein stain for 1 minto check the quality of transfer and then the membrane was blocked usingamber blocking solution (4.5% v/v teleostean gelatin, 2% w/v non-fatmilk powder, 0.1% w/v Tween-20 in Tris-buffered saline, pH 7.5) for 2hours followed by overnight incubation (12-15 hours at 4° C. on constantspeed shaker) with appropriately diluted antibodies. For Flag detection,we have used 2000-fold diluted anti-Flag M2 Antibody (Sigma), for His-6detection, we have used 500-fold anti-His6 antibody (Santa-Cruz Biotech,CA.) and for c-Myc detection, we have used 500-fold diluted anti-C-Mycantibody (Santa-Cruz Biotech, CA.).

After primary antibody incubation, the membrane was washed with TBST(Tris-buffered saline, pH 7.5 with 0.1% Tween-20) four times (10 mineach wash) and incubated with appropriately diluted secondary antibodies(10,000-fold diluted) for 1 hour at room temperature on constant speedshaker. The unbound secondary antibodies were washed with TBST (4washes/10 min each) and the blot was visualized using an ECL-Pluschemiluminescence detection system (Amersham-Pharmacia Biotech, NJ).

The results are shown in FIGS. 30A and 30B. FIG. 30A shows the totalprotein stain of PVDF membranes following protein transfer from the gelfor three replicate blots containing a minus DNA negative control and aplus p53 DNA sample respectively. FIG. 30B shows the same blots (totalprotein staining is reversible) are probed with antibodies against thethree epitope tags using standard chemiluminescent Western blottingtechniques. Arrows indicate the position of p53.

EXAMPLE 24 Gel-Based PTT for Cancer Genes

The detection of truncating mutations in proteins was first reported byRoest and co-workers and applied to the detection of truncatingmutations in the APC gene by Vogelstein, Kinzler and co-workers.Truncations in a translated protein can occur due to frameshift,splicing and point mutations which result in the occurrence of a stopcodon in the reading frame of a gene. Truncated polypeptides can bedetected by translating a specific region of the DNA corresponding tothe target gene in an in vitro system in the presence of radioactivelabels (e.g. ³⁵S-methionine) and then analyzing the resultingpolypeptide using standard PAGE. Such an approach has been reported forthe analysis of truncating mutations in a variety of cancer-linked genesincluding BRCA1/BRCA2, ATM, MHS2, MLH1. However, the use of radioactiveisotopes presents problems in terms of the time needed for detection (>5hours), which is critical for high-throughput analysis. For this reason,it would be highly advantageous to replace radioactivity with a morerapid means of detection.

In this example, we demonstrate the feasibility of rapid truncationanalysis based on the use of N-terminal tags. The present inventionprovides a convenient, accurate and rapid method to screen fortruncation mutations in a wide range of genes of clinical significance.The Example involves 1) PCR with primers having sequences complementaryto the APC gene, 2) preparation of the tRNA, 3) in vitro translation, 4)detection of nascent protein.

1. PCR of Clinical Samples

Clinical samples were submitted to the Washington University MolecularDiagnostics laboratory for screening of chain truncations in the APCgene, which are characteristic of the autosomal dominant cancer syndromefamilial adenomatous polyposis (FAP). Genomic DNA was isolated and aspecific region of the APC gene (Exon 15-segment 2) was first amplifiedby PCR using primers which incorporate a T7 promoter, and Kozak sequenceinto the DNA. The forward Primer, T7-APC2 was5′-GGATCCTAATACGACTCACTATAGGGAGA CCACCATGGATGCATGTGGA-ACTTTGTGG-3′ (SEQID NO:18) and the reverse primer 3′-APC2 was5′-GAGGATCCATTAGAT-GAAGGTGTGGACG-3′ (SEQ ID NO:19). The underlinedsequence in forward primer is T7 promoter and the sequence shown initalics corresponds to that of Kozak sequence which is necessary forefficient eukaryotic translation initiation. The PCR reaction mixture of50 μl contained 200-500 ng template DNA (either WT or mutant), 0.5 μMeach primer and 25 μl of PCR master mix (Qiagen, CA.) and nuclease freewater (Sigma Chemicals, St. Louis. Mo.) water. The PCR was carried outusing Hybaid Omni-E thermocyler (Hybaid, Franklin, Mass.) fitted withhot-lid following conditions: 95° C. for 3 min, followed by 40 cyclesconsisted of 95° C. for 45 sec. 55° C. for 1 min and 72° C. for 2 minand the final extension at 72° C. for 7 min. The PCR product wasanalyzed on 1% agarose gel and the PCR amplified DNA was used in thetranslation reaction without any further purification.

2. Preparation of the tRNA

The purified tRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was firstamino-acylated with lysine. The typical aminoacylation reaction (100 μl)contained 1500 picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HClbuffer, pH 7.5, 10 mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl andexcess of aminoacyl tRNA-synthetases (Sigma). The reaction mixture wasincubated for 45 min at 37° C. After incubation, the reaction mixturewas neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 andsubjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5volumes) was added to the aqueous phase and the tRNA pellet obtained wasdissolved in the water (35 μl). To this solution 5 ul of 0.5M CAPSbuffer, pH 10.5 was added (final concentration of 50 mM) followed by 10ul of 10 mM solution of BODIPY-FL-SSE. The mixture was incubating for 10minutes at 0° C. and the reaction was quenched by the addition of freelysine (final concentration=100 mM). To the resulting solution 0.1volumes of 3 M NAOAc (pH=5.0) was added and the modified tRNA wasprecipitated with 3 volumes of ethanol. Precipitate was dissolved in 50μl of RNase-free water and passed through Sephadex G-25 gel filtrationcolumn (0.5×5 cm) to remove any free fluorescent reagent, if present.The modified tRNA was stored frozen (−70° C.) in small aliquots in orderto avoid free-thaws. The modification extent of the aminoacylated-tRNAwas assessed by acid-urea gel electrophoresis (Varshney, U., Lee, C. P.& RajBhandary, U. L., 1991 J. Biol. Chem. 266, 24712-24718).

3. Translation

The PCR products (see step 1 above) were directly added withoutpurification to a small aliquot of a Promega rabbit reticulocyte TnTQuick system which also contained the BODIPY-Lys-tRNA (see step 2above). More specifically, after PCR, 0.5-1 μl of PCR product wasdirectly added to translation reaction mixture containing 8 μl of rabbitreticulocyte extract for PCR product (Promega), 0.5 μl of 1 mM completeamino acid mix, 1 μl of BODIPY-FL-Lysyl-tRNA. The translation reactionwas allowed to proceed for 1 hour and the reaction product were analyzedby 14% SDS-PAGE. Imaging was performed in under 1-2 minute using aMolecular Dynamics FluorImager.

4. Detection

FIG. 31 shows the results for analysis of several different humangenomic samples using BODIPY-FL-lysyl-tRNA^(lys) to incorporate afluorescent label into fragments of the APC protein. Lane 1 is a minusDNA control. Lane 2 shows the results for wild-type DNA, while lanes 3-8show the results for various mutant DNA isolated from patients havingFAP (colon cancer). The last lane is fluorescent molecular weightmarkers. As seen in FIG. 31, the WT DNA (lane 2) produces a band, whichcorresponds to the normal Exon 15, segment 2 fragment of the APC gene.In contrast, all other lanes (except lane 6) exhibit the WT band and anadditional band which corresponds to truncated fragments of Exon 15,segment 2. Thus, these individuals are heterozygous and carry one WT andone chain truncating mutation in the APC gene. In contrast, the lane 6results indicates normal WT sequence in this region for both genes.Similar conclusion was reached independently using conventionalradioactive PAGE analysis of patient samples by the University MolecularDiagnostics laboratory.

A similar analysis was performed to detect chain-truncating mutations inExon 15-segment 3 (FIG. 32). Proteins were synthesized using the rabbitreticulocyte in vitro translation system in conjunction withBODIPY-FL-lysyl-tRNA^(lys). Following separation by SDS-PAGE, translatedproteins were visualized by fluorescence imaging to (FIG. 32A) or bychemiluminescent Western blotting procedures using a polyclonal antibodydirected against the BODIPY fluorophore (FIG. 32B). Lane 1 is a minusDNA control. Lane 2 shows the results for APC3 wild-type DNA, while lane3 shows the results for APC3 truncated mutant. Lane 4 shows the resultsfor APC2 wild-type DNA, while lane 5 shows the results for APC2heterozygous mutant.

Overall, these results demonstrate the ability to replace radioactivePTT screening with fluorescent-based screening of chain truncationsinvolved in human inherited diseases.

EXAMPLE 25 Gel-Free PTT for Cancer Genes

Although the replacement of radioactivity in Example 24 (above) withfluorescent labels represents an improvement in current PTT technology,it still relies on the use of gels, which are not easily adaptable forhigh-throughput screening applications. For this reason, this exampledemonstrates a non-gel approach based on the use of chemiluminescentdetection. In this approach, a cancer-linked protein or polypeptidefragment from the protein is expressed in vitro from the correspondinggene with different detection and binding tags incorporated at theN-terminal, C-terminal and between the two ends of the protein using acombination of specially designed primers and tRNAs. The detection andbinding tags provide a means to quantitate the fraction of protein orprotein fragment which is truncated while the tags located between thetwo ends of the protein can be used to determine the region oftruncation. For example, a full-length protein would contain both an Nand C-terminal tag, whereas a truncated protein would contain only theN-terminal tag. The signal from a tag incorporated at random lysinesbetween the two ends of the protein (intrachain signal) would be reducedproportional to the size of the truncated fragment. It is important toalso capture the protein with a marker located close to the N-terminusin order to avoid interference of chain truncations with binding.

In order to evaluate this method, we performed experiments on the APCand p53 genes containing either a WT sequence or truncating mutations.In both cases, a combination of primers and specially designed tRNAswere used to incorporate a series of markers into the target proteinsduring their in vitro synthesis in a rabbit reticulocyte system. Afterin vitro expression, the expressed protein was captured in 96-well ELISAplates using an affinity element bound to the plate. The relative amountof N-terminal, C-terminal and intrachain signal was then determinedusing separate chemiluminescent-based assays.

1. PCR of Cancer Genes

A. APC Segment 3

First, the genomic DNA (WT and isolated from cell lines harboring mutantAPC gene) was amplified by PCR using following primers. The forwardprimer, PTT-T7-APC3, was5′-GGATCCTAATACGACTCACACTATAGGGAGACCACCATG-CACCACCATCACCATCACGGAGGAGATTACAAAGATGACGATGACAAA-GTTTCTCCATACAGGTCACGGGGAGCCAAT-3′(SEQ ID NO:20) and the reverse primer, PTT-Myc-APC3, was5′-ATTATTACAAATCCTCTTCCGAGATTAA-TTTTTGTTCACTTCTGCCTTCTGTAGGAATGGTATCTCG-3′(SEQ ID NO:21). The underlined sequence in forward primer is T7promoter, nucleotides shown in italics corresponds to that of His-6 tagwhile the nucleotides sequence shown in the bold codes for FLAG-epitopeand the rest of the primer is the complementary region for APC segment 3DNA. In the reverse primer, the underlined sequence corresponds to thatof c-Myc epitope. The PCR\reaction mixture of 50 μl contained 200-500 ngtemplate DNA (either WT or mutant), 0.5 μM each primer and 25 μl of PCRmaster mix (Qiagen, CA.) and nuclease free water (Sigma Chemicals, St.Louis, Mo.) water. The PCR was carried out using Hybaid Omni-Ethermocyler (Hybaid, Franklin, Mass.) fitted with hot-lid usingfollowing conditions: 95° C. for 3 min, followed by 40 cycles consistingof 95° C. for 45 sec, 55° C. for 1 min and 72° C. for 2 min and thefinal extension at 72° C. for 7 min. The PCR product was analyzed on 1%agarose gel and the PCR amplified DNA was used in the translationreaction without any further purification.

B. P53

The p53 DNA was prepared as described in Example 23 (above).

2. Preparation of the tRNA

The BODIPY-FL-lysyl-tRNA^(lys) was prepared as described in Example 23(above). Preparation of Biotin-lysyl-tRNA^(lys) andPC-Biotin-lysyl-tRNA^(lys) was achieved as follows. The purifiedtRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was first aminoacylatedwith lysine. The typical aminoacylation reaction contained 1500picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HCl buffer, pH 7.5, 10mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl and excess ofaminoacyl-tRNA-synthetases (Sigma Chemicals, St. Louis, Mo.). Thereaction mixture was incubated for 45 min at 37° C. After incubation,the reaction mixture was neutralized by adding 0.1 volume of 3 M sodiumacetate, pH 5.0 and subjected to chloroform:acid phenol extraction(1:1). Ethanol (2.5 volumes) was added to the aqueous phase and the tRNApellet obtained was dissolved in water (35 μl). To this solution 5 μl of0.5 M CAPS buffer, pH 10.5 was added (50 mM final conc.) followed by 10μl of 10 mM solution of either Biotin or photocleavable-Biotin. Themixture was incubated for 10 min at 0° C. and the reaction was quenchedby the addition of lysine (final concentration=100 mM). To the resultingsolution 0.1 volume of 3 M NaOAc, pH=5.0 was added and the modified tRNAwas precipitated with 3 volumes of ethanol. Precipitate was dissolved in50 ul of water and purified on Sephadex G-25 gel filtration column(0.5×5 cm) to remove any free fluorescent reagent, if present. Themodified tRNA was stored frozen (−70° C.) in small aliquots in order toavoid free-thaws. The modification extent of the aminoacylated-tRNA wasassessed by acid-urea gel electrophoresis (Varshney, U., Lee, C. P. &RajBhandary, U. L., 1991, J. Biol. Chem. 266, 2471224718).

3. Translation

The typical translation reaction mixture (20 μl) contained 16 μl of TNTrabbit reticulocyte extract for PCR DNA (Promega, Madison, Wis.), 1 μlof amino acid mix (1 mM), 1-2 μl of PCR DNA (see APC and p53 preparationdescribed above) and RNase-free water. For fluorescence detection, theBODIPY-FL-lysyl-tRNA^(lys) was included into the translation reactionmixture. The translation reaction was allowed to proceed for 60 min at30° C.

4. Detection

FIG. 33A shows the results of an initial experiment designed to detect achain truncation introduced into the p53 protein during RT-PCR In thiscase an N-terminal FLAG epitope was used for capture (see thedescription of the capture assay using 96-well ELISA plates at thebeginning of the EXPERIMENTAL section), and His₆ and c-myc used for theN- and C-terminal markers, respectively. Detection of the N-terminusHis-tag was achieved using a peroxidase labeled nickel chelate-baseddetection probe (India™ His Probe-HRP, Pierce, Rockford, Ill.).Detection of the C-terminus was performed using a rabbit polyclonalantibody directed against the human c-myc epitope followed by aperoxidase labeled mouse anti-[rabbit IgG] secondary antibody. As seen,the ratio of C/N terminal signals is reduced approximately 25-fold forthe truncated protein compared to WT. Further optimization of this assayshould result in sensitivity sufficient to detect truncating mutationsin 1/100 mutant/WT p53 proteins, thus enabling applications tonon-invasive colon cancer screening.

In a second experiment (FIG. 33B), capture was facilitated with anN-terminal His₆-tag, while FLAG and c-myc were used as N and C-terminalmarkers, respectively. In addition, an intrachain photocleavable biotinmarker was incorporated by adding PC-Biotin-Lys-tRNA to the in vitromixture. Biotin detection was achieved using peroxidase labeledNeutrAvidin™ (Pierce). The results show a 13-fold reduction in the C/Nratio for truncated p53 compared to WT. Furthermore, the intrachainbiotin signal drops by 75% relative to the N-terminal signal.

A third chemiluminescent protein truncation assay was designed to detectchain truncation in the APC gene of a mutant cell line (FIG. 33C).Capture was facilitated with an N-terminal His₆-tag, while FLAG andc-myc were used as N and C-terminal markers, respectively. As seen inFIG. 33C, the truncated APC exhibits a marked drop in the C/N ratio (⅙)again indicating the presence of a chain truncation.

Overall, these experiments demonstrate the ability to detectchain-truncating mutations in cancer-linked proteins using a gel-freechemiluminescent approach.

EXAMPLE 26 Fluorescent Immunoprecipitation Method

In this Example, the use of Fluor-tag tRNA in immunoprecipitation isdemonstrated. In this experiment, once the nascent protein wassynthesized in the presence of the labeled tRNA, it was captured onprotein-G beads using specific antibodies. All other (unbound) materialis removed. The bound protein of interest is then released from the beadby SDS-treatment and analyzed by PAGE.

The in vitro translation was carried out using either the PCR DNA codingfor P53 protein or a plasmid DNA coding for α-hemolysin (prepared asdescribed in previous examples). After the translation reaction, equalamounts of 10% SDS (w/v) was added to the reaction mixture and thesamples were boiled for 5 min. The samples were diluted with I buffer(Tris-buffer saline with 1% Triton X-100) and 2 μg of either anti-P53antibody (Santa Cruz Biotech, CA) or anti-HL antibody (Sigma, St. Louis,Mo.) was added and incubated for several hours at 4° C. with mixing. Asa negative control, immunoprecipitation was carried out usingnon-specific mouse IgG antibodies. After antibody incubation, Protein-Gbeads (Pierce, Ill.) were added to the reaction mixture and furtherincubated for 1 hour with constant mixing. The beads were then pelletedand washed three time with TBS with 0.1% SDS. Finally, the beads weresuspended into 50 μl of 2×SDS-gel loading buffer and boiled for 5 min.The SDS-PAGE was carried out according to Laemmli (Laemmli, U. K. 1970,Nature, 227, 680-685).

The gel was then scanned using FluorImager S1 (Molecular Dymanics.Sunnyvale, Calif.) equipped with argon laser as excitation source. Forvisualization of BODIPY-FL labeled nascent protein, 488 nm was used asexcitation source as it is the closest to its excitation maximum and foremission (we have used 530±30 filter). The gel was scanned using PMTvoltage 1000 volts and either 100 or 200 micron pixel size.

FIG. 34 shows the results. Lane M contains fluorescent markers. Lane 1is a minus DNA control (+anti-HL antibody). Lane 2 shows the results forHL DNA+mouse IgG. Lane 3 shows the results for HL DNA+anti-HL antibody.Lane 4 is a minus DNA control (+anti-P53 antibody). Lane 5 shows theresults for p53 DNA+mouse IgG. Lane 6 shows the results for p53DNA+anti-p53 antibody. It should be clear that the immunoprecipitationmethod provides specific results and a very clean signal without anysubstantial background.

EXAMPLE 26 Suppressor tRNAs

When using BODIPY-labeled initiator tRNAs, a competition occurs betweenendogenous initiator tRNA (unlabeled) which causes reduced incorporationof the label. To overcome this competition, the present inventioncontemplates using an amber stop codon and a corresponding suppressortRNA.

1. Preparation of BODIPY-Val-pdCpA

The first step involves the preparation of a BODIPY-amino acid conjugatefor attachment to the suppressor tRNA. The synthesis is set forth inFIG. 35. The synthesis begins with the preparation of5-Methyl-1(2-Nitrophenyl)ethanol (compound 2). To begin,5-Methyl-2-nitroacetophenone (Olejnik, J., S. Sonar, E.Krzymanska-Olejnik, and K. J. Rothschild. 1995 Proc Natl Acad Sci USA.92:7590-4; Olejnik, J., E. Krzymanska-Olejnik, and K. J. Rothschild.1998. Methods Enzymol. 291:135-54.) was dissolved in 5 ml of ethanol. Tothis solution 5 mg of sodium borohydride was added in small portionsuntil analytical TLC showed complete conversion to5-Methyl-1(2-Nitrophenyl)ethanol. The reaction was quenched by additionof 5 ml acetone and acidified to pH=3.0 with 1 N HCl, followed byextraction with chloroform. The organic layer was dried and evaporatedto give the target compound as yellowish oil. This compound was usedwithout any further purification.

Continuing with the synthesis scheme, 5-Methyl-1(2-Nitrophenyl)ethanol(350 mg; 1.95 mmol) (compound 2) was dissolved in 10 ml of acetonitrile.To this solution 407 ul of triethylamine (1.5 eq) was added followed byN,N′-disuccinimidyl carbonate (750 mg, 1.5 eq.). The reaction mixturewas stirred at room temperature until analytical TLC (silica gel,chloroform) showed quantitative conversion to compound 3. The resultingsolution was added directly to the solution of triethylammonium salt ofvaline.

Valine (218 mg; 1.86 mmol) was suspended in 2 ml of water to which wasadded 260 ul of triethylamine. To this solution a solution of compound 3was added xx over 15 min. period, during which pH value of the mixturewas adjusted if necessary by the addition of triethylamine. The mixturewas stirred for 30 min and acidified by addition of 10 ml of 0.1 N HCl,followed by extraction with 3×20 ml AcOEt. Organic layers were combined,dried and evaporated to dryness. The crude product was dissolved in 20ml chloroform and extracted with 4×30 ml of 0.1 N NaHCO3, the aqueouslayer acidified and re-extracted by chloroform to give 320 mg of puretarget Npe-Val (compound 4).

Npe-Val (compound 4) (320 mg, 1 mmol) was dissolved in 320 ul ofacetonitrile. To this solution was added triethylamine (280 ul, 2 eq.)and chloroacetonitrile (190 ul, 3 eq.). The mixture was stirredovernight at room temperature. 20 ml of methylene chloride was addedfollowed by 20 ml of 1N NaHSO4, the mixture was extracted with 4×20 mlmethylene chloride. Organics were evaporated to dryness and purified onsilica gel column using step gradient of ethyl acetate in hexane(10-50%) to give 300 mg of pure product (compound 5).

20 OD of pdCpA (Annovis) containing 2.2 equiv of tetrabutylammoniumcounterion was dissolved in 40 ul of dry DMF. To this solution was addeda solution of 2 equivalents of Npe-Val-COOCH2CN (0.66 mg) in 10 ul DMF.The reaction was kept at RT for 2 hrs, then the product was isolatedusing preparative HPLC to give 15 OD of compound 6.

15 OD₂₆₀ of Npe-Val-pdCpA was dissolved in 500 ul of 25 mM NaOAc, pH=4.5and irradiated for 20 minutes using 365 nm light (BlakRay XX-15, UVP).166 ul of BODIPY-FL-SE (@10 mg/ml; 7 equivs. was added) followed by 140ul of 1N NaHCO₃. Additional portions of BODIPY-FL-SE solution (2×166 ul,total 21 equivs) were added after 5 and 10 minutes, respectively, andthe reaction mixture was vortexed at room temperature for 45 minutes.The product (compound 7) was isolated using preparative HPLC NovapakC18, 10×100 mm, linear gradient of acetonitrile in 50 mM TEAA, pH 5.4over 90 min., flow 2 ml/min) and characterized by absorption at both 260and 505 nm. Yield 7.25 OD₂₆₀.

2. Expression and Purification of Amber-Suppressor fMet-tRNA.

PstI fragment of nusA operon containing a gene for mutamt tRNA^(fmet)(S. Ishi et al. PNAS v.81, pp409-413, 1984) was cloned into pGEM4 vector(Promega, Madison, Wis.). Total tRNA was isolated from overnight culture(100 ml) of E. coli XL-blue 2 cells by phenol extraction and ethanolprecipitation. Suppressor tRNA^(fmet) was purified using native PAGE(Seong and RajBhandary, PNAS v. 84 pp. 334-338, 1987).

3. tRNA Truncation by Venom Phosphodiesterase I (VPD I) (FIG. 36)

1 OD₂₆₀ of purified initiator suppressor tRNA was dissolved in in 50 uLof 50 mM Tris-HCl pH 7.5;10 mM MgCl₂ and 100 mM NaCl. To this solution0.5 unit of VPD I was added and the mixture was incubated for 30 min at0° C. After phenol extraction tRNA was precipitated by EtOH, dried anddissolved in 100 uL of water. Selective (—CA) dinucleotide removal fromthe 3′-terminus was confirmed by 8%, 7M Urea PAGE analysis.

4. Ligation with BODIPY-Val-pdCpA with Truncated Initiator SuppressortRNA

As shown in the scheme of FIG. 36, the tRNA is charged with theBODIPY-amino acid conjugate in a ligation reaction. 0.5 OD₂₆₀ of tRNAwas incubated with 0.5 OD₂₆₀ unit of pdCpA in 100 ul of ligation buffer(50 mM Tris-HCl pH 7.8; 10 mM MgCl₂; 10 mM DTT; 1 mM ATP 30% of DMSO)and 200 units of T4 RNA ligase (New England Biolabs, Beverly, Mass.) for1 hour at 37° C. After the incubation tRNA was precipitated by 3-4volumes of EtOH, dissolved in 50 ul of water and spine-filtered usingG25 MicroSpin column (Amersham Pharmacia Biotech Inc.). Ligated tRNA wasaliquoted and stored at −70° C. The product will be referred to as aninitiator/amber suppressor tRNA.

5. Translation and Analysis

Translation was performed in a cell-free E. coli S30 extract (Promega,Madison, Wis.) in 20 ul total volume. The mixture contained DNA codingfor the particular protein (1 ug) and initiator amber suppressor tRNAprepared by chemical aminoacylation (1 ug). The mixture was incubatedfor 1 hour at 37° C. and then 1 ul was mixed with the loading buffer,heat denatured and run on a 14% SDS-PAGE (150 V, 1 hr). The gel was thenrinsed briefly and analyzed using Fluoroimager in case of BODIPY label.In case of PC-Biotin the gel was blotted onto PVDF membrane and thebiotin detected using streptavidin-HRP.

FIG. 37 shows the results. Lane 1 is a minus DNA control+BODIPY-Valinitiator/amber suppressor tRNA. Lane 2 shows the results for luciferaseDNA+BODIPY-Lys-tRNA. Lane 3 shows the results for amber-1 luciferaseDNA+BODIPY-Lys-tRNA, while lane 4 shows the improved results for amber-1luciferase DNA+BODIPY-Val initiator/amber suppressor tRNA.

Lane 5 shows the results for hemolysin DNA+BODIPY-Lys-tRNA. Lane 6 showsthe results for amber-1 hemolysin DNA+BODIPY-Lys-tRNA, while lane 7shows the improved results with amber-1 hemolysin DNA+BODIPY-Valinitiator/amber suppressor tRNA.

Lane 8 shows the results for DHFR DNA+BODIPY-Lys tRNA. Lane 9 shows theresults for amber-1 DHFR DNA+BODIPY-Lys tRNA, while lane 10 shows theimproved results for amber-1 DHFR DNA+BODIPY-Val initiator/ambersuppressor tRNA.

EXAMPLE 28 Drug-Proteome Screening

The completion of the human genome project has opened a new chapter indrug discovery. In contrast to conventional methods, where a druglibrary is screened against a single protein, the entire proteome is nowavailable for screening. However, such drug-proteomic screens aredifficult to accomplish using conventional technology because of theneed to rapidly convert genome to proteome, engineer specially labeledproteins and screen thousands of samples per hour.

The present invention provides methods and compositions that aim atremoving these limitations by screening drug-protein interactions usingcell-free expressed labeled proteins and 2-D microarrays. The presentinvention contemplates incorporating the above-described labels andmarkers (e.g. fluorescent and affinity tags) into proteins during theircell-free expression. Fluorescent tags (e.g. at the N-terminal,C-terminal, within and/or throughout the protein) provide a means tomonitor protein expression and detect their interactions with potentialdrug compounds. Affinity tags provide a means to rapidly isolatecell-free expressed proteins from the cell-free lysate. Drug screensperformed on 2-D microarrays of cell-free expressed proteins, reflectingall or part of the human proteome, provide a system for high sensitivitydetection, high throughput and low sample volume requirements.

The proteomic screening assays of the present invention greatlyaccelerate the drug discovery process by allowing potential interactionsbetween a lead drug compound and an entire proteome (or part of aproteome) to be screened. Such information is valuable to explore themolecular basis of a drug's action and to identify possible side effectsof a drug by constructing a drug interaction map.

A drug-interaction map provides information about the interaction of aspecific compound (e.g. a lead drug) with the human proteome. In orderto establish a drug-interaction map, methods are contemplated that allowsystematic expression and labeling of entire proteomes using a formattedcDNA library that consists of a series of plasmid cDNA pools. Each pool,consisting of approximately 50 plasmids or less (more preferablyapproximately 40 plasmids or less, still more preferably approximately30 plasmids or less, and most preferably approximately 25 plasmids orless), is simultaneously expressed in a cell-free reaction mixture (e.g.Promega TnT rabbit reticulocyte expression system). The proteins areaffinity purified from the mixture using linkers such as PC-biotinincorporated during translation with tRNAs. The expressed proteins arethen exposed to a surface (e.g. a bead or slide), which has theimmobilized test drug compound on the surface. Interaction of one ormore proteins from each pool is then detected using the incorporatedfluorescent labels. The identity of a particular protein(s) from thepool which interacts with a drug compound is then determined usingeither a procedure which subdivides the plasmid pool or throughmicroanalysis of the immobilized protein. Alternatively, the expressedproteins in each pool are transferred to a solid medium usingincorporated affinity tags. Each pool of proteins are arranged on a 2-Darray and interrogated with compounds, which are immobilized on labeledbeads. High throughput screening can be accomplished using 2-Dmicroarrays and fluorescent imagers similar to DNA-based microchips.

An important advantage of the use of cell-free expressed proteins withnon-radioactive markers, such as incorporated fluorescent and/oraffinity markers, compared to conventional methods of radioactivelabeling is the ability to perform rapid isolation of nascent proteinsand rapid detection with the sensitivity necessary for high throughputanalysis. This approach is especially advantageous in the case of 2-dmicroarrays where radioactive detection is precluded. In addition, it isimportant to limit the number of the plasmids in each pool (e.g. lessthan 50) so that each protein coded by the cDNA is expressed at asufficient level to detect. While large volumes of reaction mixture(e.g. 1 ml) can always be utilized to express pools of plasmids, largevolume reactions would be prohibitively expensive for practicalproteomic-screening. For example, utilization of 2000 1 ml reactionvolumes would be necessary to express 2000 plasmids pools. Using currentcommercial costs of TnT rabbit reticulocyte this would costapproximately $800,000. In contrast, smaller volume 50 microliterreactions compatible with 2-d microarray formats would cost only$20,000. Earlier methods of in vitro expression cloning such asdescribed in U.S. Pat. No. 5,654,150 (hereby incorporated by reference)are based on collecting pools of about 100 individual bacterial coloniesand expressing proteins encoded by the cDNAs in the pools in vitro.While this approach represents an improvement of earlier (non-in vitro)expression cloning methods such as described in U.S. Pat. No. 4,675,285(also incorporated by reference), it still does not generally provide asufficiently sensitive method to perform high throughput drug-proteomicscreening provided by the present invention. In addition todrug-proteomic screening, the present invention would also allow for therapid throughput screening of interactions of the proteome with DNA andother biomolecules.

Two different related approaches for drug-proteomic screening arecontemplated, both based on the utilization of 2-D arrays or microarraysand detection using fluorescence, luminescence, chemiluminescence,absorption or other means of electromagnetic detection (either directlyor indirectly). As shown in FIG. 38, the first approach utilizescell-free expressed proteins produced from pools of plasmids, whichcomprise a formatted genomic library. In order to eliminate interferencefrom preexisting protein in the reaction mixture, which exist at muchhigher levels than the expressed proteins, a pre-purification step maybe necessary using incorporated affinity tags and complementary capturemolecules coated on a surface. Each sample from an individual poolcontaining the expressed proteins is then applied to a spot on thesurface containing the immobilized target drug. The incorporatedfluorescence marker is then used to identify those samples, whichcontain proteins, which interact with the target drug. Alternatively, afluorescently labeled molecule (e.g. an antibody), which interacts withan incorporated affinity marker can be used. In one preferredembodiment, a biotin is used as the affinity marker, which interactswith a streptavidin-horse radish peroxidase (HRP) complex whichcatalyzes a reaction that produces a detectable signal (e.g.chemiluminescent or fluorescent).

Once a protein pool is identified which exhibits an interaction with thetarget drug through detection of fluorescence from the particular spot,the actual interacting protein can be identified using a variety ofprocedures. One method relies on the retransformation of the individualplasmids in the pool followed by in vitro expression of proteins fromeach individual plasmids in the presence of FluoroTags or Affinity Tags.An alternative approach is to recover the actual bound protein(s) from aspot showing positive fluorescence and perform sequence analysis eitherusing mass spectrometry or through chemical means.

A second related approach for drug-proteome screening is alsocontemplated (FIG. 39). In this case, the samples containing thecell-free expressed proteins produced from pools of plasmids are firstbound to surfaces and then interrogated with a specific target drug,which is conjugated to a fluorescent moiety. The incorporated affinitytags are used for the binding of the in vitro expressed protein to aspecially prepared surface containing a capture molecule which interactswith the affinity tag. Incorporated fluorophores are used to monitorsurface binding. Target drug binding is detected using dual wavelengthfluorescence detection or alternatively fluorescence resonance energytransfer, which assures that the protein and bound drug are in closeproximity. The drug-protein complex can also be released using aphotocleavable biotin linker, for downstream analysis to confirm thedrug-protein interaction and for further mass spectrometric analysis ofthe drug or protein identity.

In both scenarios described above, parallel expression of the genomiclibrary, which is estimated to consist of 2000 individual plasmid poolsof approximately 50 plasmids per pool, provides the basis to performrapid drug-proteomic analysis. For example, using a preformatted set ofreaction wells (50 microliters each) and plasmid sets, the entireparallel expression reaction could be accomplished in less than 1 hour.The purification of the expressed proteins from each sample could alsobe extremely rapid, provided that suitable equipment (e.g. 96well-pipetters operated by robotic arms) were utilized. For example,pipette filter tips can be prepared that contain an affinity media forcapture via the in vitro incorporated photocleavable tag (e.g.streptavidin bound to beads) and light can be used for subsequentrelease.

In both scenarios, the samples can be rapidly transferred to a surfacefor subsequent drug-protein fluorescent assays. For example, in the caseof slides coated with the target drug, individual samples can be spottedusing a commercial arrayer such as the Affymetrix 417 arrayer, whichoffers 125 um resolution spot size. In principle, a single microscopeslide would be sufficient to array the entire proteome with 5-foldredundancy in less than 10 minutes. Larger spots sizes or greaterredundancy would require more slides, however the entire preparation ofthe arrayed proteome would still rapid and amenable to high throughputprocessing. The entire “proteome on a slide” concept is particularlyattractive, since the rapid production of such slides would enablescreening of the entire proteome versus libraries of drugs.

For the above mentioned format, rapid read-out can be achieved using afluorescent 2-D microarray reader. For example, the Affymetrix 418scanner can scan an entire slide in approximately 10 minutes and is ableto perform 3 color imaging. Furthermore, the array reader offers highprecision (10 microns), speed (18 mm/min) and sensitivity (less than 1Cy3 dye molecules per 1 square micron with >3.5 S/N).

In order to evaluate the screening assays of the present invention,experiments were performed which involved the incorporation of PC-Biotinor BODIPY into the test protein dihydrofolate reductase (DHFR).Experiments were aimed at evaluating whether PC-Biotin or BODIPY markersincorporated into DHFR during its in vitro synthesis could be used toassay for its interaction with the small ligand methotrexate, acompetitive inhibitor for DHFR whose normal ligand is dihydrofolate.Since methotrexate can reduce the rate of cell growth, it is widely usedas an anticancer drug in chemotherapy.

In one assay, PC-Biotin was incorporated into DHFR during its in vitroexpression in S30 in the presence of PC-Biotin-Lys-tRNA. Afterexpression, the crude mixture was exposed to methotrexate-coated agarosebeads. After extensive washing, specific binding of the DHFR to thebeads was assayed using streptavidin-HRP. As seen in FIG. 40, a signalis obtained only when PC-Biotin-Lys-tRNA is included in the expressionsystem. UV-light exposure prior to binding, abolishes the signalrelative to control levels (data not shown), providing a convenientmethod to measure background levels during automated assays.

In a second experiment, capillary electrophoresis (CE-LIF) was used as ahomogenous solution assay to detect the interaction of DHFR andmethotrexate. In this case, BODIPY was incorporated at the N-terminus ofDHFR during its in vitro synthesis using BODIPY-fmet-tRNA. Theelectropherogram for free DHFR exhibited a peak at 2.9 minutes (data notshown). In comparison, the electropherogram of DHFR preincubated withmethotrexate for 30 minutes at room temperature exhibited a broadenedpeak at 2.8 min (data not shown). The change in migration time isattributed to the complex formation between DHFR and methotrexate.

In a third series of experiments, the ability to selectively bind anddetect an in vitro expressed target protein using an antibody wasexplored. For this purpose, PC-Biotin was incorporated into p53expressed in rabbit reticulocyte lysate after RT-PCR of a pooled humanmRNA sample using the PC-Biotin-Lys-tRNA. The protein was thenspecifically captured on ELISA plates using monoclonal antibody cloneBP53-12 (Santa Cruz Biotechnology, Santa Cruz, Calif.) which recognizesboth WT and mutant conformations. As a negative control, an identicalquantity of pre-immune mouse IgG was used for capture, in place of thep53 specific antibody. Detection of the biotin was achieved usingperoxidase labeled NeutrAvidin biotin binding protein (Pierce, Rockford,Ill.) in a standard ELISA assay. The results (not shown) indicated thatonly specific binding with the p53 antibody results in a significantsignal. These data demonstrate the feasibility of studying p53 bindingusing a panel of conformation specific antibodies which selectivelyreact with different p53 mutants.

EXAMPLE 29 Incorporation of VSV-G and p53-Derived Epitopes

Genomic DNA and RNA (WT and APC mutant) was isolated from establishedcell lines CaCo-2 (C1), HCT-8 (C2) and SW480 (C3) as well as frompatient blood samples using commercially available kits (Qiagen,Valencia, Calif.). PCR amplification of a selected region of the APCgene (APC segment 3) was carried out using 250-500 ng of genomic DNA,0.2 μM primer mix (forward and reverse) and 1×PCR master mix (Qiagen,Valencia, Calif.). Amplification was performed as follows: an initialcycle of denaturation at 95° C., forty cycles of denaturation at 95° C.for 45 sec. annealing at 57° C. for 45 sec, extension at 72° C. for 2min and a final extension step at 72° C. for 10 min. RT-PCRamplification of APC gene (APC segment 3) was carried out using one-stepRT-PCR/PCR kit from ClonTech (Palo Alto, Calif.). RT-PCR reactioncontained 500 ng of total RNA, 0.2 μM primer mix (forward and reverse)and 1×RT-PCR master mix. Amplification conditions were the same as abovewith an additional initial cycle of reverse transcription at 50° C. for1 hour. The primer pair was:

Forward: (SEQ ID NO:22)5′-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGGCTACACCGACAT-CGAGATGAACCGCCTGGCAAGGTTTCTCCATACAGGTCACGGGGA GCC-3′

Reverse: (SEQ ID NO:23) 5′-TTATTACAGCAGCTTGTGCAGGTCGCTGAAGGTACTTCTGCCTTCTG T-AGGAATGTATC-3′

The italicized nucleotides in the forward primer correspond to the T7promoter, the underlined ATG is the initiation codon, the boldfacenucleotide region codes for the N-terminal tag (VSV-G; YTDIEMNRLGK: SEQID NO:39) and the remaining nucleotide sequences correspond to thecomplementary region of the APC gene. In the reverse primer, theboldface nucleotides code for the C-terminal tag (P53 sequence derivedtag; TFSDLHKLL: SEQ ID NO:24) while the rest of the nucleotide sequenceis complementary to the APC gene and nucleotides in italics codes for 2successive stop codons. After amplification, the quality and quantity ofthe PCR products was analyzed by agarose gel electrophoresis.

EXAMPLE 30 Cell-Free Protein Synthesis

The cell-free reaction mixture contained 8 μl of TNT T7 Quick RabbitReticulocyte lysate for PCR DNA (Promega, Madison, Wis.), 0.5 μl of acomplete amino acid mix and 0.5 μl of DNA (approximately 200 ng) andeither 1 μl of biotin-lysyl-tRNA or a tRNA mix consisting of equalamount of Biotin-lysyl tRNA and BODIPY-FL-lysyl-tRNA. The translationreaction was allowed to proceed for 45 min at 30° C. Forelectrophoresis, a 4-6 μl aliquot was used for SDS-PAGE. SDS-PAGE wascarried out according to Laemmli. Kahmann et al., A Non-RadioactiveProtein Truncation Test For The Sensitive Detection Of All Stop AndFrameshift Mutations, Hum Mutat 19; 165-172 (2002). Afterelectrophoresis, polyacrylamide gels were scanned using a FluorImager SI(Molecular Dynamics, Sunnyvale, Calif.) equipped with an Argon laser asan excitation source (488 nm line) and a 530±30 nm emission filter.

EXAMPLE 31 High-Throughput Solid-Phase PTT (HTS-PTT)

After the translation exemplified in Example 30, the reaction mixturewas diluted 30-fold with TBS containing 0.05% Tween-20, 0.1% TritonX-100, 5% BSA, and both antibodies (anti-VSV-G-HRP (Roche AppliedSciences, Indianapolis, Ind.) at 80 ng/mL and anti-p53-alkalinephosphatase at 100 ng/mL (Santa Cruz Biotechnology, Santa Cruz,Calif.)). Subsequently, 100 μl of the diluted reaction mixture was addedto each well of a NeutrAvidin™ coated 96-well plate (pre-blocked with 5%BSA) and incubated for 45 min on an orbital shaker. NeutrAvidin™ wasobtained from Pierce Chemicals (Rockford, Ill.) and Microlite2+multiwell plates were obtained from Dynex Technologies (Chantilly, Va.).The plate was washed 5×with TBS-T (TBS with 0.05% Tween-20) followed by2× with TBS and developed using a chemiluminescent HRP substrate (SuperSignal Femto, Pierce Chemicals, Rockford, Ill.). Finally, the plate waswashed twice in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 50 mM magnesiumacetate, a chemiluminescent alkaline-phosphatase reaction mixture.

EXAMPLE 32 Comparison of tRNA^(TOTAL) Versus tRNA^(lys)

The use of biotin-lysyl-tRNA (i.e., biotin-tRNA^(lys)) to incorporatebiotin affinity tags at lysine residues would result in no capture ifthe chain truncation occurs upstream of the first lysine. This problemand the overall efficiency of capture can be improved if a tRNA mixturecontaining most, or all, of the normal cellular tRNAs ismisaminoacylated with a biotin-labeled amino acid. This “total tRNAmixture” (i.e., tRNA^(TOTAL)) is then used instead of lysyl-tRNA (i.e.,tRNA^(lys)), thereby making the biotin incorporation less dependent onthe amino acid sequence of the nascent protein.

The translation was essentially carried out as described in Example 30in the presence of biotinylated-tRNA^(lys) and biotinylated HTS-PTT.ELISA was carried out as described in Example 31. FIG. 46 depicts ELISAdata showing samples translated in presence of biotin-tRNA^(TOTAL) have2-4 times higher signals than that of samples translated in presence ofbiotin-tRNA^(lys).

EXAMPLE 33 A Penta-Lysine 5′-Tag

The use of biotin-lysyl-tRNA to incorporate biotin affinity tags atlysine residues would result in no capture if the chain truncationoccurs upstream of the first lysine. This problem and the overallefficiency of capture can be improved if an extra lysine sequence areartificially added in the beginning the transcript. This can be achievedby adding 5 extra lysine coding nucleotides in the 5′ primer after anATG codon or after the epitope coding nucleotides. This design of thetranscript then can increase the overall number of lysine residuesresulting in the increased incorporation of biotin usingbiotin-tRNA^(lys). It is contemplated that the lysine tag includes, butis not limited to, from between 3-10 lysine residues.

The translation was essentially carried out as described in Example 30.ELISA HTS-PTT was carried out as described in Example 31. FIG. 47depicts ELISA data showing increased signal in samples having extralysine residues using shorter nascent proteins (WT is 70 kD nascentprotein while is N3 is a mixture of 70 and 30 kD nascent proteins).

EXAMPLE 34 Gel Electrophoresis: APC-23 and APC-3

This example demonstrates SDS-PAGE separation of the nascent 140 kDAPC-23 protein fragment from the 70 kD APC-3 protein fragment.

The HTS-PTT is not limited by the resolution of SDS-PAGE, therefore, itis possible to reduce the number of cell-free reactions per patientsample by translating larger segments of the target gene (or the wholegene itself). The translation was essentially carried out as describedin Example 30. This study demonstrates that HTS-PTT analysis offragments of at least 140 kDa in size is possible. (See FIG. 48)

EXAMPLE 35 Comparison of APC-3 and APC-23 Template DNA

HTS-PTT was carried out using either APC-3 (1.7 kb DNA fragment) orAPC-23 (3.56 kb DNA fragment). The translation was essentially carriedout as described in Example 30. FIG. 49 shows bar graph datademonstrating that there are no significant differences in the C/N ratioirrespective of the template used. This result indicates that evenlarger templates can be used for HTS-PTT, thus reducing the overallnumber of reactions per analysis.

EXAMPLE 36 Minimal Copy PCR Amplification

This example demonstrates that a single copy DNA may be amplified andisolated using HTS-PTT.

A defined amount of genomic DNA (WT APC) and cell line DNA (mutant APC)was used as the template for PCR. Low copy PCR was carried out in twosequential PCR amplifications. To test the limit of PCR, template DNAwas diluted to various ratios to obtain 1-300,000 copies of DNA/μl. Inthe first PCR amplification, a selected region of the APC gene (APClong, 3.8 kb region) was carried out using various amounts of genomicDNA, 0.2 μM primer mix (Long 5′ and Long 3′) and 1×PCR master mix(Qiagen, Valencia, Calif.). Amplification was performed as follows: aninitial cycle of denaturation at 95° C., forty cycles of denaturation at95° C. for 45 sec, annealing at 57° C. for 45 sec, extension at 72° C.for 4 min and a final extension step at 72° C. for 10 min. The productafter this PCR was used as the template for the second PCR reaction. PCRamplification of a selected region of the APC gene (APC-3) was carriedout using 5 μl of above PCR product (after APC Long PCR), 0.2 μM primermix (forward and reverse) and 1×PCR master mix (Qiagen, Valencia,Calif.). Amplification was performed as follows: an initial cycle ofdenaturation at 95° C., forty cycles of denaturation at 95° C. for 45sec, annealing at 57° C. for 45 sec, extension at 72° C. for 4 min and afinal extension step at 72° C. for 10 min.

The primer pairs were:

-   Long 5′: 5′-TTTTTGGTTGGCACTCTTACTTACCGGAGC-3′ SEQ ID NO: 47-   Long 3′: 5′-AGATGCTTGCTGGACCTGGTCCATTATCTT-3′ SEQ ID NO: 48-   Forward: SEQ ID NO: 22-   5′-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGGCTACACCGACAT    CGAGATGAACCGCCTGGCAAGGTTTCTCCATACAGGTCACGGGGAGCC-3′-   Reverse: SEQ ID NO: 23-   5′-TTATTACAGCAGCTTGTGCAGGTCGCTGAAGGTACTTCTGCCTTCTGTA GGAATGGTATC-3′

The italicized nucleotides in the forward primer correspond to the T7promoter, the underlined ATG is the initiation codon, the boldfacenucleotide region codes for the N-terminal tag (VSV-G; YTDIEMNRLGK, SEQID NO:39) and the remaining nucleotide sequences correspond to thecomplementary region of the APC gene. In the reverse primer, theboldface nucleotides code for the C-terminal tag (P53 sequence derivedtag: TFSDLHKLL, SEQ ID NO:24). The nucleotides in italics (TTATTA) codesfor 2 successive stop codons. After amplification, the quality andquantity of the PCR products were analyzed by agarose gelelectrophoresis. FIG. 50 demonstrates that the PCR product correspondsto a single copy of DNA.

EXAMPLE 37 MicroArray-Based HTS-PTT

This example checks the feasibility of MicroArrays for PTT.Specifically, various amount of amplified WT and mutant APC DNA(cell-line) were mixed and translated. The translation was essentiallycarried out as described in Example 30.

Each reaction mixture received an equal amount of spotting buffer(Tris-buffered saline, 0.05% Tween-20 with 40% glycerol) and 0.1 μl ofreaction mixture without any pre-purification was spotted onstreptavidin-coated glass slides (pre-blocked with 5% bovine serumalbumin, BSA). After 30 min incubation at 37° C. in a humidifiedchamber, all the excess biotin-binding sites on the slides were blockedby immersing the slide into 5 mM biotin solution. Then the slide wasincubated for 45 min at room temperature with anti-P53 antibody(BP-P53-12; Santa Cruz Biotechnology, Santa Cruz, Calif.) with shaking.After removal of excess antibody from the slide, the slide was developedusing Alexa-488-conjugated anti-mouse secondary antibody (MolecularProbes, Eugene Oreg.). Visualization of the slide (see FIG. 51) shows aconcentration-dependent color intensity pattern. (Molecular DynamicsFluorImager SI.).

EXAMPLE 38 High Sensitivity Gel-Free ELISA-PTT

This example demonstrates the incorporation of reporter molecules at theposition of the mutation in order to obtain a mutation-specific positivesignal. This procedure utilizes a high sensitivity, chemiluminescent“Gel-Free” PTT for non-invasive CRC screening.

Although the HTS-PTT for inherited mutations (e.g., 50% mutant DNA and50% wild-type DNA) works well, it is insufficient for non-invasive coloncancer screening, which requires a truncating mutation detectionsensitivity of 1:100 mutant:WT-APC. To achieve this, a differentapproach is contemplated by this invention involving production of apositive signal only when a truncation mutation is encountered, insteadof attempting to measure a decrease in C-terminal signal as describedabove. FIG. 52 exemplifies the assay.

For example, α-hemolysin (HL) may be translated as a model test proteinin an E. coli cell-free translation system. Two types of DNA, wild typeDNA (WT) and mutant DNA (e.g., Amb-1) are introduced into the cell-freetranslation system. Amb-1 introduces an inappropriate/misplaced amber(UAG) stop codon into the mRNA. The DNA was mixed at various ratios ofmutant to wild-type while maintaining a constant amount of total DNA.The translation was performed with an amber suppressor tRNAmisaminoacylated with photocleavable (PC)-Biotin.

Following translation, the mutant protein is selectively captured viathe incorporated biotin moiety using NeutrAvidin™ coated 96-well ELISAplates (removal of unused biotinylated tRNA was found not to benecessary). Plates were then probed with an antibody directed againstthe HL protein and signal is generated using a typical enzyme-linkedchemiluminescence detection system. A translation reaction performedwith 100% wild-type DNA is used as the negative control (i.e., Blank) toaccount for the level of non-specific binding of HL to the ELISA plate.FIG. 53 panel A indicates that as little as 0.4% mutant (i.e., 1 mutantper 250 wild-type) can be detected with statistical significance versusthe Blank. The p value of the statistical analysis is 0.002 (n=3) basedon a two-tailed, unpaired, homoscedastic t-test and is 25-fold betterthan the 0.05 required for statistical significance. As seen in FIG. 53panel B, larger ratios of mutant to wild type are easily detectableusing this system. FIG. 54 demonstrates signal linearity with variousratios of mutant to wild-type DNA. Linear regression analysis yields alinear correlation coefficient of 0.9963 (99.63% linear correlation) forthe data points tested shows the linearity of the assay.

EXAMPLE 39 High Sensitivity Gel-Based PTT

This example demonstrates the application of a highly sensitive PTTdetection system to identify mutated DNA within a mixture of normal DNAfrom stool samples.

The gel-based PTT test is designed to detect mutant protein derived froma DNA sample containing 1 mutant gene in a pool of 100 wild-type genes.The data from this experiment using a gel-based PTT (See FIG. 55) showsthat when wild-type and mutant DNA from stool samples of an FAP patientwas mixed at various ratios, as little as 4% mutant DNA is detectable(i.e., 4 copies of mutant DNA in a pool of 96 wild-type genes). Specificembodiments of this invention contemplate improving detectionsensitivity further by affinity purification and immunoprecipitation,incorporating linkers, markers and epitopes, improving FluoroTagsensitivity and improving overall translation yields.

This protocol provides a high sensitivity gel-based fluorescent PTTanalysis of APC segment 2 (APC2) for non-invasive CRC screening.Specifically, PCR amplified DNA was derived from a clinical sample knownto be positive for an inherited (FAP) heterozygous truncation mutationin APC2. Mutant DNA was mixed at various ratios with the WT DNA and usedto perform an in vitro translation of APC2. A constant level of totalDNA was used for all translation reactions. Translations are performedusing BODIPY-FL-Lys-tRNA^(lys) to label the nascent protein. FIG. 55panel A shows the fluorescently imaged gel following SDS-PAGE. Lane“−DNA” (i.e., minus DNA) corresponds to a translation reaction performedwhere only the DNA was omitted. The six middle lanes each contain adifferent percentage of mutant DNA (i.e., 0% is pure WT DNA), while laneM corresponds to 100% mutant (heterozygote) DNA. The asterisk located atthe bottom of the 4% lane indicates the lowest level of mutant DNA thatcan be detected reliably with the current technique. The arrow locatedat the bottom left corner indicates the position of the truncated APC-2fragment. FIG. 55 Panel B depicts quantification of the bandingintensity for the truncated APC-2 fragment (see arrow) bycomputer-assisted densitometry. The integrated intensity values isplotted versus the percent of mutant DNA (lowest point plotted is 4%mutant DNA). Linear regression analysis solved the line equation andlinear correlation coefficient are shown on the graph.

EXAMPLE 40 MALDI-Mass Spectrometry (MALDI-MS) Mutation Detection

This example utilizes α-HL with a C-terminal His6-tag, which wasexpressed in a S30 reaction mixture using a high-expression plasmidcontaining the α-HL gene under control of the T7 promoter.

For comparison, a mutant of α-HL (S302W) was also expressed in E. colitranslation extract. In both cases, the proteins were isolated from thetranslation reaction mixture using Co²⁺-NTA chromatography. The isolatedprotein was dialyzed, concentrated and deposited on a MALDI substrate.As seen in FIG. 57, a peak is observed at 34,884 Da for WT α-HL and34,982 Da for the mutant α-HL, in good agreement with calculated masses(34,890 and 34,989 Da, respectively). This demonstrates the ability ofMALDI to detect a mutation in an in vitro expressed protein.

EXAMPLE 41 Improved Signal-to-Noise Ratios

This example demonstrates an improved method of ELISA antibody-baseddetection following HTS-PTT. The translation and HTS-PTT was essentiallycarried out as described in Examples 30 and 31, respectively.

The data shown in FIG. 58 indicates that using enzymes directlyconjugated to the primary antibody (i.e., HRP or AP) improves the signalto noise ratio relative to the traditional combination of primaryantibodies and enzyme-conjugated secondary antibodies. A directcomparison of the signals and backgrounds obtained using anti-VSVprimary followed by secondary HRP-conjugated antibody as well as usingHRP-conjugated anti-VSV primary antibody demonstrates a greater signalfor the directly conjugated VSV-HRP. Similar analysis was also carriedout using anti-P53 antibodies (data not shown).

EXAMPLE 42 Single-Step ELISA

This example demonstrates a shortened ELISA protocol to reduce overallanalysis time. The translation and HTS-PTT was essentially carried outas described in Examples 30 and 31, respectively.

The traditional ELISA process includes putting the nascent protein(translation extract) onto plate, washing of the plate, antibodyincubation, washing of the plate and then development of signals. In theshortened protocol, however, the antibody is mixed with nascent protein(i.e., translation extract) prior to putting onto the plate. After theextract-antibody incubation, this mixture was put on the plate andincubated. This protocol consolidates the steps of binding of theantibodies to nascent protein and binding of the nascentprotein-antibody complexes to the plate. This consolidation does notresult in any significant difference in signal or C/N ratio obtained forN-terminal and C-terminal by using either method. (See FIG. 59)

EXAMPLE 43 Simultaneous Single-Well C-& N-Terminal Signal Detection

This example provides one embodiment of HTS-PTT that contemplates thedetection of N-terminal and C-terminal signals in a single well (i.e.,from the same sample). The translation and HTS-PTT was essentiallycarried out as described in Examples 30 and 31, respectively.

Traditionally, HTS-PTT samples are first divided and subjected toN-terminal and C-terminal detection using two different antibodies inseparate wells. This step assumes that the biotin-binding capability ofthe nascent protein does not vary from well to well. Although thisassumption may be correct, detection of N- and C-terminals in the samewell on the same sample reduces the overall risk of error.

This simultaneous assay uses two differentially conjugated antibodies inthe same well and sequentially measures their respective signals. Inthis particular assay, the N-terminal is marked using HRP-conjugatedanti-VSV antibody and the C-terminal is marked using AP-conjugatedanti-P53 antibody. (See FIG. 60) After the antibody incubation, the HRPsignal was read. Then wells were washed with 100 mM Tris-HCl, pH 9.5containing 100 mM NaCl and 50 mM magnesium acetate to completely inhibitHRP activity thus forming an optimum environment to detect AP activityusing appropriate substrates. The data clearly shows that simultaneousincubation and sequential signal detection provided equivalent data asthe more traditional separate-well assay.

EXAMPLE 44 HTS-PTT High Sensitivity

This example demonstrates the high sensitivity of the ELISA-PTT assaycontemplated by one embodiment of this invention. The translation andHTS-PTT was essentially carried out as described in Examples 30 and 31,respectively.

FIG. 61 shows signal intensity using various amounts of translationextracts of N-terminal, C-terminal signals and signal-to-noise ratios(i.e., 0.0 translation/well). Very low amounts are detectable (i.e., 0.1ul of translation mix per well) when compared against a signal-to-noiseratio of approximately 10 and 30 for N- and C-terminal, respectively.

EXAMPLE 45 Enzyme Substrate Discovery

This example demonstrates that the technology contemplated by thisinvention comprising the incorporation of N-terminal and C-terminal tagsinto nascent protein can be used very effectively for discovery of thesubstrates for enzymes. The translation was carried out as described inExample 30. Two examples of these enzymes include, but are not limitedto, proteases and kinases. In one embodiment, Caspase-3 degraded N- andC-terminally tagged MDM and APC protein in a time-dependent manner,while similarly tagged PKA was unaffected. (see FIG. 63). Concurrently,the time-dependent appearance of the digestion product is shown in FIG.64. Similar results were also obtained for several other proteinsexpressed in vitro in presence of either FluoroTag (gel-based) orbiotin-tRNA (ELISA-based)(data not shown). It is contemplated that anyenzyme resulting in a physical alteration of the substrate is capable ofbeing monitored using this assay system.

EXAMPLE 46 Enzyme Discovery

This method demonstrates an ability for one embodiment of the presentinvention to label a plasmid with a marker protein. The translation wasdescribed as carried out in Example 30.

For example, multiple plasmid pools in vitro rabbit reticulocytetranslation extracts are able to express a variety of protein/proteinfragments. After doping one of the plasmid pools with the small amountof the plasmid coding for enzyme luciferase this specific expressionvector was subsequently identified. Following transformation, thenascent proteins was translated in presence of biotin-tRNA andsubsequently captured on streptavidin-coated plate. After washingunbound protein, the doped plasmid pool was identified by detectingluciferase activity using a chemiluminescent substrate and plate reader.(See FIG. 65). Similar methods can be used for kinase enzyme discovery.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered exemplary only, with the scope of particularembodiments of the invention indicated by the following claims.

Name and Molecular weight Formula Fluorescence Properties BODIPY-FL,SSEM. WT. 491

Excitation = 502 nmEmmision = 510 nmExtinction = 75,000 NBDM. WT. 391

Excitation = 466 nmEmmision = 535 nmExtinction = 22,000 Bodipy-TMR-X,SEM. WT. 608

Excitation = 544 nmEmmision = 570 nmExtinction = 56,000 Bodipy-R8GM. WT.437

Excitation = 528 nmEmmision = 547 nmExtinction = 70,000 Fluorescein(FAM)M. WT. 473

Excitation = 495 nmEmmision = 520 nmExtinction = 74,000 Fluorescein(SFX)M. WT. 587

Excitation = 494 nmEmmision = 520 nmExtinction = 73,000 PyMPOM. WT. 582

Excitation = 415 nmEmmision = 570 nmExtinction = 26,000 5/6-TAMRAM. WT.528

Excitation = 546 nmEmmision = 576 nmExtinction = 95,000

TABLE 3 −FluoroTag ™ tRNA +FluoroTag ™ tRNA Enzyme/Protein Translationreaction Translation reaction α-Hemolysin 0.085 0.083 OD_(415nm)/μlLuciferase 79052 78842 RLU/μl DHFR 0.050 0.064 ΔOD_(339nm)/μl

TABLE 4 Various Epitopes and Their Sequences Amino acid Name sequenceNucleotide sequence (5′→ 3′) His-6 HHHHHH CATCACCATCACCATCAC (SEQ IDNO:62) (SEQ ID NO:61) FLAG DYKDDDDK GACTACAAGGACGACGACGACAAG (SEQ IDNO:64) (SEQ ID NO:63) c-Myc EQKLISEEDL GAGCAGAAGCTGATCAGCGAGGAGGACCTG(SEQ ID NO:66) (SEQ ID NO:65) Strep- WSHPQFEK TGGAGCCACCCCCAGTTCGAGAAG(SEQ ID NO:68) Tag (SEQ ID NO:67) Amber- CSPFEVQVSPEATGCAGCCCCTTCGAGGTGCAGGTGAGCCCCGAGGCCGGCGCCCAGAAG 16 GAQK (SEQ ID NO:70)(SEQ ID NO:69) T7-Tag MASMTGGQQMG ATGGCCAGCATGACCGGCGGCCAGCAGATGGGC (SEQID NO:72) (SEQ ID NO:71) VSV-G^(#-) YTDIEMNRLGKTACACCGACATCGAGATGAACCGCCTGGGCAAG (SEQ ID NO:74) Tag (SEQ ID NO:73)HA*-Tag YPYDVPDYA TACCCCTACGACGTGCCCGACTACGCC (SEQ ID NO:76) (SEQ IDNO:75) Protein- EDQVDPRLIDGK GAGGACCAGGTGGACCCCCGCCTGATCGACGGCAAG C Tag(SEQ ID NO:77) (SEQ ID NO:78) HSV^($-) QPELAPEDPEDCAGCCCGAGCTGGCCCCCGAGGACCCCGAGGAC Tag (SEQ ID NO:79) (SEQ ID NO:80)^(#)Vesicular Stomatitis Virus Glycoprotein *HemAgglutinin ^($)HerpesSimplex Virus glycoprotein

1. An oligonucleotide having the sequence set forth in SEQ ID NO:
 22. 2.A kit comprising the oligonucleotide of claim
 1. 3. The kit of claim 2,further comprising a polymerase.